Journal: Stem cells (Dayton, Ohio)

Article Title: Conversion of human umbilical cord mesenchymal stem cells in Wharton's jelly to dopaminergic neurons in vitro: potential therapeutic application for Parkinsonism.

doi: 10.1634/stemcells.2005-0053

Figure Lengend Snippet: Figure 1. The grouping and scheme of in vitro incubation of HUMSCs. Tyrosine hydroxylase–positive populations were gener- ated from undifferentiated HUMSCs by a three-step in vitro differ- entiation method. Abbreviations: DMEM, Dulbecco’s modified Ea- gle’s medium; FBS, fetal bovine serum; FGF, fibroblast growth factor; HUMSC, human umbilical mesenchymal stem cell; NCM, neuronal-conditioned medium; Shh, sonic hedgehog.

Article Snippet: In stage 3, cells were supplemented with NCM or 10% FBS-DMEM in the presence of the murine N-terminal fragment of sonic hedgehog (SHH) (500 ng/ml, 461-SH, R&D Systems Inc., Minneapolis, http://www.rndsystems.com) and murine FGF8 isoform b (FGF8) (100 ng/ml, 423-F8, R&D Systems Inc.) for 3, 6, 9, or 12 days.

Techniques: In Vitro, Incubation, Modification

Journal: Stem cells (Dayton, Ohio)

Article Title: Conversion of human umbilical cord mesenchymal stem cells in Wharton's jelly to dopaminergic neurons in vitro: potential therapeutic application for Parkinsonism.

doi: 10.1634/stemcells.2005-0053

Figure Lengend Snippet: Figure 2. HUMSC differentiation into dopaminergic, norepinephrine, and GABAergic neurons in vitro. (A): Photomicrographs showing TH immunocytochemistry of cultured HUMSCs. The cells expressed TH after incubation with NCM for 6 days and then SHH and FGF8 in DMEM for 3 days. In addition to TH-positive neurons, DBH-positive (B) and GAD-positive (C) neurons were detected. Human-specific nuclear antigen are in green, and DBH and GAD are in red. Arrows indicate cells stained positively for TH, DBH, or GAD. Scale bar 100 m. (D): Histograms showing the percentage of TH-positive cells after incubation with NCM, SHH, and FGF8. (Results represent the mean standard error from three different experiments. At least 200 cells were counted from 10 randomly selected microscopic fields in each experiment. Statistics consisted of one-way ANOVA followed by the LSD test; *statistical difference at p .05 compared with NCM-only group.) (E): TH expression in cultured cells by Western blotting. The molecular weight of rat and human TH were 60 and 68 kDa, respectively. Rat SN served as positive control. (F): Dopamine concentration in culture medium after HUMSCs were treated with NCM, SHH, and FGF8. (Results represent the mean standard error from three different experiments. Statistics consisted of one-way ANOVA followed by the LSD test; *statistical significance at p .05 compared with DMEM and NCM-only groups.) Abbreviations: ANOVA, analysis of variance; DBH, dopamine--hydroxylase; DMEM, Dulbecco’s modified Eagle’s medium; FGF, fibroblast growth factor; LSD, least-significant difference; HUMSC, human umbilical mesenchymal stem cell; NCM, neuronal-conditioned medium; Shh, sonic hedgehog; SN, substantia nigra; TH, tyrosine hydroxylase.

Article Snippet: In stage 3, cells were supplemented with NCM or 10% FBS-DMEM in the presence of the murine N-terminal fragment of sonic hedgehog (SHH) (500 ng/ml, 461-SH, R&D Systems Inc., Minneapolis, http://www.rndsystems.com) and murine FGF8 isoform b (FGF8) (100 ng/ml, 423-F8, R&D Systems Inc.) for 3, 6, 9, or 12 days.

Techniques: In Vitro, Immunocytochemistry, Cell Culture, Incubation, Staining, Expressing, Western Blot, Molecular Weight, Positive Control, Concentration Assay, Modification

Journal: Stem cells (Dayton, Ohio)

Article Title: Conversion of human umbilical cord mesenchymal stem cells in Wharton's jelly to dopaminergic neurons in vitro: potential therapeutic application for Parkinsonism.

doi: 10.1634/stemcells.2005-0053

Figure Lengend Snippet: Figure 5. Rotation behavior in response to amphetamine tested at 1, 2, 3, 4, and 5 months after lesion. A significant decrease in the number of amphetamine-induced turning was seen in animals with grafted cells treated with NCM SHH FGF8 (, n 6) compared with control (lesion-only) animals (F, n 12) and lesioned animals that received grafted cells treated with NCM (E, n 12). Statistics consisted of two-way ANOVA followed by the LSD test. (* Significant difference at p .05 between NCM SHH FGF8-treated group compared with the control and NCM groups at the same time point. # Significant difference at p 0.05 between the control and NCM groups over 1-month intervals.) Abbreviations: ANOVA, analysis of variance; FGF, fibroblast growth factor; LSD, least-significant difference; NCM, neuronal- conditioned medium; Shh, sonic hedgehog.

Article Snippet: In stage 3, cells were supplemented with NCM or 10% FBS-DMEM in the presence of the murine N-terminal fragment of sonic hedgehog (SHH) (500 ng/ml, 461-SH, R&D Systems Inc., Minneapolis, http://www.rndsystems.com) and murine FGF8 isoform b (FGF8) (100 ng/ml, 423-F8, R&D Systems Inc.) for 3, 6, 9, or 12 days.

Techniques: Control

Journal: bioRxiv

Article Title: Loss of Kallmann syndrome-associated gene WDR11 disrupts primordial germ cell development by affecting canonical and non-canonical Hedgehog signalling

doi: 10.1101/2020.09.06.284927

Figure Lengend Snippet: (A) Immunofluorescence analyses of Ptch2, Gas1, Boc and p-Src on the GR sections of WT and Wdr11 KO embryos at E10.5. PGCs are labelled by SSEA1 staining. The merged images are shown without DAPI signal to improve the clarity. Scale bar, 10μm. (B) The relative fluorescence intensity values of Ptch2, Gas1, Boc and p-Src, that were normalised with the fluorescence intensity values of SSEA1 in each cell. Data are obtained from WT (n=8), KO (n=10) for Ptch2; WT (n=10), KO (n=12) for Gas1; WT (n=9), KO (n=11) for Boc; WT (n=11), KO (n=12) for p-Src. Error bars represent means ± SD. Statistical analysis by unpaired t-test with Welch’s correction. ****P < 0.0001. (C) PGCs in the GR primary cultures generated from E10.5 embryos of WT and Wdr11 KO were analysed after immunofluorescence co-staining of p-Creb and SSEA1. Cells plated on 0.1% gelatin-coated cover slips were treated with solvent dimethyl formamide (DMF) or recombinant Shh protein (Shh-N) for 10 minutes. Representative images are shown. Scale bar, 10μm. (D) The relative fluorescence intensity values of p-Creb normalized with the intensity values of SSEA1 observed in each PGC were compared in each of the genotype group, with or without Shh-N treatment for 10 minutes. Data are obtained from DMF (n=8) and Shh-N (n=9) for WT; DMF (n=9) and Shh-N (n=9) for Wdr11 KO. Error bars represent means ± SD. Statistical analysis by unpaired t-test with Welch’s correction. ****P < 0.0001.

Article Snippet: Cells were incubated in 0.5% serum-containing media before treatment with 200 ng/mL recombinant Shh N-terminal peptide (R&D Systems, 1314-SH) diluted in dimethyl formamide (DMF).

Techniques: Immunofluorescence, Staining, Fluorescence, Generated, Solvent, Recombinant

Journal: The Journal of Clinical Investigation

Article Title: Sonic Hedgehog repression underlies gigaxonin mutation–induced motor deficits in giant axonal neuropathy

doi: 10.1172/JCI129788

Figure Lengend Snippet: (A) NIH-3T3 cells were transfected with human Cherry-Gig plasmid ± 3 μg/mL Shh for 48 hours. Degradation of endogenous Ptch protein occurs only in the presence of both ectopic gigaxonin and Shh. (B) Repression of the endogenous gigaxonin using siRNA causes an increase of the endogenous Ptch levels in Shh Light-2 cells, which is potentiated by Shh induction. (C and D) Interaction between gigaxonin and Ptch, as revealed by reverse immunoprecipitation on both endogenous (C) and ectopic (D) Ptch protein. Shh Light-2 cells transfected with human Cherry-Gig (C); COS-7 cells transfected with zebrafish 3Flag-Gig and zebrafish Cherry-Ptch (D). IgG serves as an internal negative control. (D) Cherry immunoprecipitation identifies Ptch proteins, which are mainly not modified, while the Ptch pool enriched in gigaxonin immunocomplex presents a laddering overlapping with K48-specific ubiquitin chain. (E) Model of action of the gigaxonin–E3 ligase in the initiation of Shh signaling. In an off state (left), prior to Shh activation, receiving cells silence the cascade through the inhibitory effect of the Ptch receptor on the effector Smo. Upon Shh production, the active Shh form is released and received by progenitor cells. Gigaxonin acts as an initiator of signal transduction by degrading Shh-bound Ptch receptor (middle), hence allowing the derepression of the signal transducer Smo, which translocates into the cilium to activate the pathway. In the absence of gigaxonin (right), receiving tissues are unable to interpret Shh signaling, due to the constitutive repression of Smo induced by the accumulation of Shh-bound Ptch receptor.

Article Snippet: At 24 hours after transfection, NIH-3T3 cells were treated with 3 μg/mL ShhN (1314-SH, R&D Systems) ( 39 ) and Light-2 cells with Shh-conditioned medium (Shh-CM) obtained from ShhN-expressing HEK293 cells for 48 hours (provided by P. Beachy, GRCF Biorepository & Cell Center, Johns Hopkins University, Baltimore, Maryland, USA) ( 64 ).

Techniques: Transfection, Plasmid Preparation, Immunoprecipitation, Negative Control, Modification, Activation Assay, Transduction