Journal: Stem cell research

Article Title: Generation of a heterozygous knockout human embryonic stem cell line for the OCIAD1 locus using CRISPR/CAS9 mediated targeting: BJNhem20-OCIAD1-CRISPR-39.

doi: 10.1016/j.scr.2015.12.037

Figure Lengend Snippet: Fig. 1. (A) Strategy for targeting OCIAD1 exon3 by CRISPR-CAS9. (B) Genomic targeting of OCIAD1 exon3 of BJNhem20-OCIAD1-CRISPR-39 identified by T7 endonuclease mediated digestion of high fidelity exon 3 amplicon detecting deletions in this clone. (C) Chromatograms showing sequence analysis to confirm heterozygous mutation in the targeted region of OCIAD1. (D) Western blot analysis showing decreased OCIAD1 levels in BJNhem20-OCIAD1-CRISPR-39. Graph represents standard error mean after analysis of three biological replicates using single factor ANOVA (P value 0.001). (E) Karyotype analysis of BJNhem20-OCIAD1-CRISPR-39. (F) Analysis of transcript levels of pluripotency marker genes Oct4, Nanog, TDGF and Sox2 by reverse transcription and polymerase chain reaction (RT-PCR) amplification. GAPDH was used to normalize transcript levels. (G) Immunostaining analysis of BJNhem20-OCIAD1-CRISPR-39 for depletion of OCIAD1 and for pluripotency markers Oct4, SSEA4 and TRA1-81 as indicated, compared to parental hESC line BJNhem20. (H) Differentiation analysis of BJNhem20-OCIAD1-CRIPSR-39: embryoid bodies stained to show differentiation to all the three germ layers by immunostaining for AFP, Brachyury and β-III tubulin marking the endoderm, mesoderm and ectoderm respectively.

Article Snippet: Primary antibodies used were against OCIAD1 (Abcam Ab91574), Oct4 (BD Biosciences BD611203), TRA1-81 and SSEA4 (kind gift from Peter Andrews, University of Sheffield, UK), Brachyury (Santa Cruz Biotech Cat no. SC-17743), β-III tubulin (Santacruz SC-51670), AFP (Sigma Chemical Pvt.

Techniques: CRISPR, Amplification, Sequencing, Mutagenesis, Western Blot, Marker, Reverse Transcription, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Immunostaining, Staining

Journal: Neurology Research International

Article Title: Subarachnoid Transplant of the Human Neuronal hNT2.19 Serotonergic Cell Line Attenuates Behavioral Hypersensitivity without Affecting Motor Dysfunction after Severe Contusive Spinal Cord Injury

doi: 10.1155/2011/891605

Figure Lengend Snippet: Neural and human markers with differentiation of the hNT2.19 cell line in vitro. The hNT2.19 cell line was treated for two weeks with retinoic acid and mitotic inhibitors and lifted to substrate-coated 8-well plastic TC slides for differentiation and immunohistochemistry for neuron-specific markers. As soon as 4 days in vitro, a variety of neural markers appeared, which remained strong until at least 6 wks of differentiation: TuJ1 (a), hNSE (b), NFL (c), NFM (d), and NFH (e). For comparison, the negative control hNT2.6 cell line was cultured similarly as the hNT2.19 cells and is here stained for TuJ1 (f). Magnification bar = 20 nm, (a–f).

Article Snippet: For immunohistochemistry of sectioned spinal cord tissues, the polyclonal antibody anti-5HT (ab10385; dilution 1/100 (in vivo)) was purchased from Abcam Inc, Cambridge, MA, and the antihuman TuJ1 antibody (Neuron-specific class III beta-tubulin) was purchased from Neuromics, Edina, MN (MO15013; dilution 1/100 (in vivo).

Techniques: In Vitro, Immunohistochemistry, Comparison, Negative Control, Cell Culture, Staining

Journal: Neurology Research International

Article Title: Subarachnoid Transplant of the Human Neuronal hNT2.19 Serotonergic Cell Line Attenuates Behavioral Hypersensitivity without Affecting Motor Dysfunction after Severe Contusive Spinal Cord Injury

doi: 10.1155/2011/891605

Figure Lengend Snippet: Transplant of hNT2.19 and hNT2.6 cell lines in the severe contusive SCI model: TuJ1 and 5HT immunohistochemistry. Rats were injured with severe contusive SCI followed at two weeks by hNT2.6 (a, b) or hNT2.19 (c, d) cell grafts. Sagittal spinal cord sections were examined at 8 wks after SCI for evidence of surviving lumbar subarachnoid hNT2.6 (a, b) or hNT2.19 (c, d) cell line grafts, utilizing TuJ1 (a, c) or 5HT (b, d) immunohistochemistry. The hNT2.19 and control hNT2.6 (10 6 cells/injection), which had been differentiated for two weeks in vitro, were injected into the subarachnoid space two weeks after the SCI. Cell graft sites were colocalized with 5HT (b, d) and the human-specific marker TUJ1 (neuron-specific class III β -tubulin; (a, c)). There are many surviving hNT2.19 (c) and hNT2.6 (a) grafted cells visible on the pial surface, which stain for TuJ1 (arrows) at the end of the experiment, 56 days after SCI and about 6 weeks after cell transplant. Adjacent sections with the same grafted hNT2.19 (d) and hNT2.6 cells (b) are stained for 5HT, but only the hNT2.19 cells (d) are labeled for 5HT (arrows).

Article Snippet: For immunohistochemistry of sectioned spinal cord tissues, the polyclonal antibody anti-5HT (ab10385; dilution 1/100 (in vivo)) was purchased from Abcam Inc, Cambridge, MA, and the antihuman TuJ1 antibody (Neuron-specific class III beta-tubulin) was purchased from Neuromics, Edina, MN (MO15013; dilution 1/100 (in vivo).

Techniques: Immunohistochemistry, Control, Injection, In Vitro, Marker, Staining, Labeling

Journal: CNS Neuroscience & Therapeutics

Article Title: TTBK2‐Driven Ciliogenesis Is Required for Intrinsic Neuronal Regeneration After Spinal Cord Injury

doi: 10.1002/cns.70763

Figure Lengend Snippet: TTBK2 regulates primary cilium formation and axonal growth in spinal neurons. (A) Schematic diagram of the primary cilium. Kinesin‐2 comprises KIF3A; TTBK2 is related to the formation of basal bodies. (B, C) Quantitative RT‐qPCR analysis showing efficient knockdown or overexpression of KIF3A and TTBK2 in spinal neurons via adenoviral infection ( n = 6 from 3 independent experiments). (D, E) Representative immunofluorescence images of spinal neurons labeled with MAP2 (green), ACIII (red), and DAPI (blue) in five experimental groups: NC, shKIF3A, shTTBK2, TTBK2‐OE, and shKIF3A + TTBK2‐OE. White arrows indicate PCs. Compared with that in NC, the cilium length was significantly reduced in the shKIF3A, shTTBK2, and shKIF3A + TTBK2‐OE groups, while TTBK2‐OE overexpression led to elongated cilia ( n = 6 from 3 independent experiments). Scale bars, 20 μm. (F, G) Representative images showing immunolabeling of TUJ1 (green, axons), MAP2 (orange, dendrites), and DAPI (blue, nuclei). Axonal morphology and length were assessed across five groups. KIF3A or TTBK2 knockdown significantly reduced axon length, while TTBK2 overexpression enhanced elongation. shKIF3A + TTBK2‐OE partially rescued axon length compared with that under shKIF3A alone ( n = 5 from 3 independent experiments). Scale bars, 10 μm. Data are presented as mean ± SEM. One‐way ANOVA was performed. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The antibodies were rabbit anti‐GFAP (Abcam, ab7260, 1:5000) mouse anti‐beta III Tubulin (TUJ1) (Proteintech, 66375‐1‐Ig, 1:400), chicken anti‐MAP2 (MAP2) (Abcam, ab5392, 1:1000), rabbit anti‐Calb (Abcam, ab108404, 1:150), mouse monoclonal antibody to ACIII (AC3) (Encorbio, MCA‐1A12, 1:1000), chicken anti‐choline acetyltransferase antibody (ChAT) (Sigma‐Aldrich, AB15468, 1:1000), rabbit anti‐TTBK2 (Sigma‐Aldrich, AB805274 , 1:1000), rabbit anti‐neurofilament‐H (NF200) (Cell Signaling, 30564, 1:400), mouse monoclonal [Rat‐401] to nestin‐neural stem cell marker (Nestin) (Abcam, ab6142, 1:1000), rabbit anti‐PSD95 (Cell Signaling, 3450, 1:400), chicken anti‐GAP43 polyclonal antibody (Thermo Fisher Scientific, PA5‐95660, 1:500), goat anti‐chicken secondary antibody goat anti‐chicken IgY H&L (Alexa Fluor 555) (Abcam, ab150170, 1:2000), goat anti‐rabbit IgG (H + L) (Alexa Fluor 647) (Beyotime, A0468 1:200), goat anti‐rabbit IgG (H + L) (Alexa Fluor 350) (Beyotime, A0408, 1:200), and goat anti‐mouse IgG (H + L) (Alexa Fluor 647) (Beyotime, A0473, 1:200).

Techniques: Quantitative RT-PCR, Knockdown, Over Expression, Infection, Immunofluorescence, Labeling, Immunolabeling

Journal: CNS Neuroscience & Therapeutics

Article Title: TTBK2‐Driven Ciliogenesis Is Required for Intrinsic Neuronal Regeneration After Spinal Cord Injury

doi: 10.1002/cns.70763

Figure Lengend Snippet: TTBK2 regulates axonal regeneration via the primary cilium–SHH pathway. (A–C) Volcano plots showing differentially expressed proteins between shTTBK2 vs. NC, shKIF3A vs. NC, and shKIF3A + TTBK2‐OE vs. shKIF3A. The x ‐axis represents log2 (fold change), and the y ‐axis represents −log10( p ‐value). Gray dots indicate proteins that did not meet significance thresholds ( p > 0.05). Blue and red dots indicate downregulated and upregulated proteins, respectively. (D) The heatmap displays differentially expressed proteins identified in each of the four experimental groups relative to the NC control group, with color intensity representing expression levels. (E–H) Western blotting analysis demonstrating significant reductions in MAP2, Gli1, and Smo protein expression in the shTTBK2 group ( n = 3 from 3 independent experiments). (I, J) Treatment with the SHH pathway agonist SAG restored MAP2 expression in shTTBK2 neurons ( n = 3, from 3 independent experiments * p < 0.05). (K, L) Representative immunofluorescence images of spinal neurons stained for TUJ1 (green), PSD95 (red), and DAPI (blue). TTBK2‐OE group showed markedly increased PSD95 expression compared to other groups ( n = 6 from 3 independent experiments). Scale bar, 5 μm. Data are presented as mean ± SEM. One‐way ANOVA was used for statistical analysis. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The antibodies were rabbit anti‐GFAP (Abcam, ab7260, 1:5000) mouse anti‐beta III Tubulin (TUJ1) (Proteintech, 66375‐1‐Ig, 1:400), chicken anti‐MAP2 (MAP2) (Abcam, ab5392, 1:1000), rabbit anti‐Calb (Abcam, ab108404, 1:150), mouse monoclonal antibody to ACIII (AC3) (Encorbio, MCA‐1A12, 1:1000), chicken anti‐choline acetyltransferase antibody (ChAT) (Sigma‐Aldrich, AB15468, 1:1000), rabbit anti‐TTBK2 (Sigma‐Aldrich, AB805274 , 1:1000), rabbit anti‐neurofilament‐H (NF200) (Cell Signaling, 30564, 1:400), mouse monoclonal [Rat‐401] to nestin‐neural stem cell marker (Nestin) (Abcam, ab6142, 1:1000), rabbit anti‐PSD95 (Cell Signaling, 3450, 1:400), chicken anti‐GAP43 polyclonal antibody (Thermo Fisher Scientific, PA5‐95660, 1:500), goat anti‐chicken secondary antibody goat anti‐chicken IgY H&L (Alexa Fluor 555) (Abcam, ab150170, 1:2000), goat anti‐rabbit IgG (H + L) (Alexa Fluor 647) (Beyotime, A0468 1:200), goat anti‐rabbit IgG (H + L) (Alexa Fluor 350) (Beyotime, A0408, 1:200), and goat anti‐mouse IgG (H + L) (Alexa Fluor 647) (Beyotime, A0473, 1:200).

Techniques: Control, Expressing, Western Blot, Immunofluorescence, Staining

Journal: CNS Neuroscience & Therapeutics

Article Title: TTBK2‐Driven Ciliogenesis Is Required for Intrinsic Neuronal Regeneration After Spinal Cord Injury

doi: 10.1002/cns.70763

Figure Lengend Snippet: The TTBK2–SHH–MAP2 axis regulates endogenous neuronal repair following SCI. (A) Representative immunofluorescence images of frozen spinal cord sections stained with GFAP (green), MAP2 (orange), TUJ1 (red), and DAPI (blue). Scale bars: Left, 200 μm; right, 50 μm ( n = 6 from six mice in each group). (B–D) The quantification of immunostaining showed increased GFAP in all injured groups. MAP2 levels in WT‐SCI were comparable to those in uninjured controls, while Ttbk2 fl/fl ‐SCI showed a marked reduction. TUJ1 staining indicated significantly higher immature neuron proportion in WT‐SCI than in other groups ( n = 6 from six mice in each group). (E, F) Co‐staining of NF200 (green) and Nestin (red) revealed elevated neural progenitor marker Nestin in injured groups. Ttbk2 fl/fl ‐SCI mice exhibited a higher Nestin/NF200 ratio than did WT‐SCI mice ( n = 6 from six mice in each group). (G–J) Western blotting results confirmed that MAP2, Smo, and Gli1 protein levels were significantly reduced in Ttbk2 fl/fl ‐SCI mice, indicating SHH pathway suppression ( n = 3 from three mice in each group). Bars and errors represent mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001 (one‐way ANOVA).

Article Snippet: The antibodies were rabbit anti‐GFAP (Abcam, ab7260, 1:5000) mouse anti‐beta III Tubulin (TUJ1) (Proteintech, 66375‐1‐Ig, 1:400), chicken anti‐MAP2 (MAP2) (Abcam, ab5392, 1:1000), rabbit anti‐Calb (Abcam, ab108404, 1:150), mouse monoclonal antibody to ACIII (AC3) (Encorbio, MCA‐1A12, 1:1000), chicken anti‐choline acetyltransferase antibody (ChAT) (Sigma‐Aldrich, AB15468, 1:1000), rabbit anti‐TTBK2 (Sigma‐Aldrich, AB805274 , 1:1000), rabbit anti‐neurofilament‐H (NF200) (Cell Signaling, 30564, 1:400), mouse monoclonal [Rat‐401] to nestin‐neural stem cell marker (Nestin) (Abcam, ab6142, 1:1000), rabbit anti‐PSD95 (Cell Signaling, 3450, 1:400), chicken anti‐GAP43 polyclonal antibody (Thermo Fisher Scientific, PA5‐95660, 1:500), goat anti‐chicken secondary antibody goat anti‐chicken IgY H&L (Alexa Fluor 555) (Abcam, ab150170, 1:2000), goat anti‐rabbit IgG (H + L) (Alexa Fluor 647) (Beyotime, A0468 1:200), goat anti‐rabbit IgG (H + L) (Alexa Fluor 350) (Beyotime, A0408, 1:200), and goat anti‐mouse IgG (H + L) (Alexa Fluor 647) (Beyotime, A0473, 1:200).

Techniques: Immunofluorescence, Staining, Immunostaining, Marker, Western Blot

Journal: Molecular Biology of the Cell

Article Title: PAG1 directs SRC-family kinase intracellular localization to mediate receptor tyrosine kinase-induced differentiation

doi: 10.1091/mbc.E20-02-0135

Figure Lengend Snippet: PAG1 TM- prevented differentiation in SH-SY5Y cells. (A) Neurite lengths of WT and PAG1 TM- SH-SY5Y cells after growth in control conditions (RPMI 1640, 2% FBS) and in differentiation conditions (RPMI 1640, 2% FBS, 10 µM RA, 5 nM NGF) * p < 0.05, n = 3. (B) Representative images of neurites after 8 d of growth are in the indicated conditions, 20× magnification. (C) Flow cytometry of β-III tubulin expression, a marker of neuronal differentiation. (D) Cell cycle analysis of WT SH-SY5Y and SH-SY5Y PAG1 TM- cells by flow cytometry. Cells were seeded in standard growth medium (RPMI 1640, 10% FBS) on collagen-coated plates and were exposed for 96 h to 10 µm RA and 5 nM NGF in low serum media (2% FBS). Cells were then stained with Hoechst 33342 and relative DNA content was measured by flow cytometry. (E) The percentage of cells in each stage of the cell cycle for each condition in D. (Results in B–D are representative of at least three independent experiments.)

Article Snippet: Flow cytometry: anti-Human/Mouse phospho-SRC (Y418) PerCP-eFluor 710 (Affymetrix/eBioscience); AF647 Mouse anti-Src (pY418) (BD Biosciences); Hoechst 33342 (CST #4082); β-III tubulin (CST #4466); pAKT-PE (pT308) (BD Biosciences); Rabbit anti- pERK-AF488 (Phospho-p44/42 MAPK [Erk1/2] [Thr202/Tyr204]) (CST #13214); Mouse IgG2b K Isotype Control PerCP-eFluor 710 (Affymetrix/eBioscience); Rabbit IgG Isotype Control AF488 (CST #4340); PE Mouse IgG1, κ Isotype Control (BioLegend #400111); AF647 Mouse IgG1, κ Isotype Control (BioLegend #400135).

Techniques: Control, Flow Cytometry, Expressing, Marker, Cell Cycle Assay, Staining

Journal: eLife

Article Title: Quantitative transportomics identifies Kif5a as a major regulator of neurodegeneration

doi: 10.7554/eLife.68148

Figure Lengend Snippet: ( a ) Optic nerves were collected 2 weeks after intravitreal injection of adeno-associated virus (AAV)-Kif5a-FLAG. Immunoprecipitation showed the presence of FLAG-tagged protein at the molecular weight of Kif5a. Co-immunoprecipitation of β-III tubulin confirmed that overexpressed Kif5a transported to the optic nerve and bound to the cytoskeleton. ( b ) RGC survival was not affected 4 weeks after viral injection of Kif5a compared to a control GFP virus (control n = 6, OE n = 7). Two-tailed, two-sample, t-test. ( c ) Representative example of wholemount retinas stained with RBPMS, an RGC-specific marker, 2 weeks after optic nerve crush, injected with either AAV-GFP or AAV-Kif5a-FLAG 2 weeks before crush. Scale bar, 100 µm. ( d ) Quantification of RBPMS + cell density after optic nerve crush (ONC) across entire retinal surface. Each point represents one retina (control n = 7, OE n = 8). Two-sample, two-tailed t-tests, p < 0.0001. Figure 7—source data 1. Raw western blots for .

Article Snippet: The membranes were saturated with TBS 1×, 0.05% Tween-20, and 5% nonfat dry milk for 1 hr at room temperature, then incubated overnight at 4°C with rabbit anti-Kif5a (1:1000, Abcam, ab5628), rabbit anti-Kif5c (1:1000, Abcam, ab192883), goat anti-biotin (1:1000, Thermo, 31852), rabbit anti-FLAG (1:2000, CST, 14,793S), rabbit anti-β-III tubulin (1:2000, 5568S, CST), rabbit anti-GAPDH (1:2000, CST, 2118), rabbit anti-SNCB (1:1000, Sigma), rabbit anti-GAP43 (1:5000, Novus), rabbit-ARF3 (1:1000, Abcam).

Techniques: Injection, Virus, Immunoprecipitation, Molecular Weight, Control, Two Tailed Test, Staining, Marker, Western Blot

Journal: bioRxiv

Article Title: OrganoLabeling: Quick and accurate annotation tool for organoid images

doi: 10.1101/2024.04.16.589852

Figure Lengend Snippet: Immunochemical characterization of embryoid bodies and 30 days old brain organoids generated under three different conditions. A) Embryoid bodies stained to demonstrate the presence of three distinct germ layers; with α-SMA (mesodermal marker), Nestin (ectodermal marker), Sox17 (endodermal marker) and DAPI (cell nucleus), B) brain organoid generated under static condition stained with Sox2 and Tuj1, C) brain organoid generated under static condition stained with N-cadherin, D) brain organoid generated using orbital shaker stained with Sox2 and Tuj1, E) brain organoid generated using orbital shaker stained with N-cadherin, F) brain organoid generated using microfluidic chip stained with Sox2 and Tuj1, G) brain organoid generated using microfluidic chip stained with N-cadherin. (Right panels; magnification of dashed zones)

Article Snippet: Subsequently, primary antibodies (α-SMA (Cell Signaling Technology, Cat no.48938S), Nestin (Proteintech, Cat no. 19483-1-AP), Sox17 (Abcam, Cat no. Ab84990), Sox2 (Bioss, Cat no. bs-0523R), Tuj1 (R&D Systems, Cat no. MAB1195), N-cadherin (Cell Signaling Technology, Cat no. 13116T) were added according to related samples and incubated for overnight at 4 °C.

Techniques: Generated, Staining, Marker

Journal: Scientific Reports

Article Title: 3D bio-printed scaffold-free nerve constructs with human gingiva-derived mesenchymal stem cells promote rat facial nerve regeneration

doi: 10.1038/s41598-018-24888-w

Figure Lengend Snippet: Differentiation of GMSC spheroids into Schwann and neuronal cells under 2D-induction conditions. ( A ) GMSC spheroid cells (spheroids) showed increased expression of Nestin, a neural progenitor cell (NPC) marker, as compared with the adherent counterparts (GMSCs). ( B ) GMSC spheroid cells were seeded onto poly-D-lysine pre-coated 4-well chamber slides and cultured under Schwann cell differentiation conditions for 14 days. The expression of S-100β and Nestin was determined by immunofluorescence studies. ( C ) GMSC spheroid cells (spheroids) and the adherent counterparts (GMSCs) were seeded onto poly-D-lysine pre-coated 4-well chamber slides and cultured under Schwann cell differentiation conditions for 14 days, and the expression of S-100β was determined by immunofluorescence studies. ( D ) GMSC spheroid cells (spheroids) and the adherent counterparts (GMSCs) were seeded onto poly-D-lysine pre-coated 4-well chamber slides and cultured under neuronal cell differentiation conditions for 14 days, and the expression of β-tubulin III was determined by immunofluorescence studies. Cell nuclei were counter-stained by DAPI (blue). Scale bar: 20 µm. Data are representative of 3 independent experiments.

Article Snippet: Cultured cells fixed with 4% paraformaldehyde (PFA) or cryosections of GMSC spheroids were blocked and permeabilized for 1 h at room temperature in PBS with 2.5% goat serum and 0.5%Triton X‐100, followed by incubation with the following primary antibodies at the appropriate dilution overnight at 4 °C: Nestin (mouse IgG, 1:250) (EMD Millipore, Burlington, MA, USA), CD29 (mouse IgG, 1:250) (BD Bioscience, San Jose, CA, USA), cleaved caspase-3 (Rabbit IgG, 1:250) (EMD Millipore), type I collagen (rabbit IgG, 1:250) (Rockland Biotech, Limerick, PA, USA), CD73(mouse IgG, 1:250) (BD Bioscience), CD90 (mouse IgG, 1:250) (BD Bioscience), vimentin (rabbit IgG, 1:250) (Boster Biological Tech., Pleasanton, CA, USA), fibronectin (rabbit IgG, 1:200 (Sigma, St. Louis, MO, USA), laminin 1 (rabbit IgG, 1:200) (EMD Millipore), β-tubulin III (mouse IgG, 1:200) (BioRad, Hercules, CA, USA), and S-100β (rabbit IgG, 1:250) (Boster Biological Tech).

Techniques: Expressing, Marker, Cell Culture, Cell Differentiation, Immunofluorescence, Staining

Journal: Scientific Reports

Article Title: 3D bio-printed scaffold-free nerve constructs with human gingiva-derived mesenchymal stem cells promote rat facial nerve regeneration

doi: 10.1038/s41598-018-24888-w

Figure Lengend Snippet: Differentiation of GMSC spheroids into Schwann and neuronal cells under 3D-induction conditions. ( A ) GMSCs or GMSC spheroids stably transduced with a Lentiviral-GFP vector were mixed with bovine type I collagen gel and filled into an Axoguard® nerve protector (3-mm diameter) and cultured in α-MEM containing 10% FBS and antibiotics for 7 days and observed under a fluorescence microscope. ( B ) GMSC spheroid cells (Spheroids) or the adherent counterparts (GMSCs) were mixed with bovine type I collagen gel and filled into an Axoguard® nerve protector (3-mm diameter) and cultured in Schwann cell differentiation medium for 14 days. The structure was fixed in 4% paraformaldehyde and cryosections were cut for immunofluorescence staining of S-100β (green) and β-tubulin III (red). ( C ) GMSC spheroid cells (Spheroids) or the adherent counterparts (GMSCs) were mixed with bovine type I collagen gel and filled into an Axoguard® nerve protector (3-mm diameter) and cultured in neuronal cell differentiation medium for 14 days. The structure was fixed in 4% paraformaldehyde and cryosections were cut for immunofluorescence staining of S-100β (green) and β-tubulin III (red). Cell nuclei were counter-stained by DAPI (blue). Scale bar: 100 µm. Data are representative of 2 independent experiments.

Article Snippet: Cultured cells fixed with 4% paraformaldehyde (PFA) or cryosections of GMSC spheroids were blocked and permeabilized for 1 h at room temperature in PBS with 2.5% goat serum and 0.5%Triton X‐100, followed by incubation with the following primary antibodies at the appropriate dilution overnight at 4 °C: Nestin (mouse IgG, 1:250) (EMD Millipore, Burlington, MA, USA), CD29 (mouse IgG, 1:250) (BD Bioscience, San Jose, CA, USA), cleaved caspase-3 (Rabbit IgG, 1:250) (EMD Millipore), type I collagen (rabbit IgG, 1:250) (Rockland Biotech, Limerick, PA, USA), CD73(mouse IgG, 1:250) (BD Bioscience), CD90 (mouse IgG, 1:250) (BD Bioscience), vimentin (rabbit IgG, 1:250) (Boster Biological Tech., Pleasanton, CA, USA), fibronectin (rabbit IgG, 1:200 (Sigma, St. Louis, MO, USA), laminin 1 (rabbit IgG, 1:200) (EMD Millipore), β-tubulin III (mouse IgG, 1:200) (BioRad, Hercules, CA, USA), and S-100β (rabbit IgG, 1:250) (Boster Biological Tech).

Techniques: Stable Transfection, Transduction, Plasmid Preparation, Cell Culture, Fluorescence, Microscopy, Cell Differentiation, Immunofluorescence, Staining

Journal: Scientific Reports

Article Title: 3D bio-printed scaffold-free nerve constructs with human gingiva-derived mesenchymal stem cells promote rat facial nerve regeneration

doi: 10.1038/s41598-018-24888-w

Figure Lengend Snippet: 3D bio-printing scaffold-free nerve constructs from GMSC spheroids. ( A ) Procedures for 3D bio-printing scaffold-free nerve constructs from GMSC spheroids. ( B ) Longitudinal section of 3D bio-printed nerve constructs. H&E staining (the left panel); immunofluorescence staining with β-tubulin III and S-100β primary antibodies. ( C ) Transverse section of 3D bio-printed nerve constructs. H&E staining (the left panel); immunofluorescence staining with β-tubulin III and S-100β primary antibodies. Cell nuclei were counter-stained by DAPI (blue). Scale bars: 200 µm. Data are representative of 2 independent experiments.

Article Snippet: Cultured cells fixed with 4% paraformaldehyde (PFA) or cryosections of GMSC spheroids were blocked and permeabilized for 1 h at room temperature in PBS with 2.5% goat serum and 0.5%Triton X‐100, followed by incubation with the following primary antibodies at the appropriate dilution overnight at 4 °C: Nestin (mouse IgG, 1:250) (EMD Millipore, Burlington, MA, USA), CD29 (mouse IgG, 1:250) (BD Bioscience, San Jose, CA, USA), cleaved caspase-3 (Rabbit IgG, 1:250) (EMD Millipore), type I collagen (rabbit IgG, 1:250) (Rockland Biotech, Limerick, PA, USA), CD73(mouse IgG, 1:250) (BD Bioscience), CD90 (mouse IgG, 1:250) (BD Bioscience), vimentin (rabbit IgG, 1:250) (Boster Biological Tech., Pleasanton, CA, USA), fibronectin (rabbit IgG, 1:200 (Sigma, St. Louis, MO, USA), laminin 1 (rabbit IgG, 1:200) (EMD Millipore), β-tubulin III (mouse IgG, 1:200) (BioRad, Hercules, CA, USA), and S-100β (rabbit IgG, 1:250) (Boster Biological Tech).

Techniques: Construct, Staining, Immunofluorescence

Journal: Scientific Reports

Article Title: 3D bio-printed scaffold-free nerve constructs with human gingiva-derived mesenchymal stem cells promote rat facial nerve regeneration

doi: 10.1038/s41598-018-24888-w

Figure Lengend Snippet: Histological analysis of newly regenerated rat facial nerves. ( A ) Left panels, H & E staining of cryosections of regenerated facial nerves; Immunohistochemistry showed increased expression of β-tubulin III and organized axonal alignment in regenerated nerves from 3D bio-printed construct transplantation as compared with silicon tube transplantation. ( B ) Immunohistochemistry showed increased expression of S-100β in regenerated facial nerves from the group with 3D bio-printed construct transplantation as compared with silicon tube control group. Scale bar: 200 µm. Cell nuclei were counter-stained by DAPI (blue). ( C ) Immunostaining of matured axonal and myelin markers, SMI31/32 and Fluoromyelin, with cryosections of regenerated facial nerves from rats transplanted with 3D bio-printed nerve grafts.

Article Snippet: Cultured cells fixed with 4% paraformaldehyde (PFA) or cryosections of GMSC spheroids were blocked and permeabilized for 1 h at room temperature in PBS with 2.5% goat serum and 0.5%Triton X‐100, followed by incubation with the following primary antibodies at the appropriate dilution overnight at 4 °C: Nestin (mouse IgG, 1:250) (EMD Millipore, Burlington, MA, USA), CD29 (mouse IgG, 1:250) (BD Bioscience, San Jose, CA, USA), cleaved caspase-3 (Rabbit IgG, 1:250) (EMD Millipore), type I collagen (rabbit IgG, 1:250) (Rockland Biotech, Limerick, PA, USA), CD73(mouse IgG, 1:250) (BD Bioscience), CD90 (mouse IgG, 1:250) (BD Bioscience), vimentin (rabbit IgG, 1:250) (Boster Biological Tech., Pleasanton, CA, USA), fibronectin (rabbit IgG, 1:200 (Sigma, St. Louis, MO, USA), laminin 1 (rabbit IgG, 1:200) (EMD Millipore), β-tubulin III (mouse IgG, 1:200) (BioRad, Hercules, CA, USA), and S-100β (rabbit IgG, 1:250) (Boster Biological Tech).

Techniques: Staining, Immunohistochemistry, Expressing, Construct, Transplantation Assay, Control, Immunostaining