Journal: Developmental cell

Article Title: Isl1 regulation of Nkx2.1 in the early foregut epithelium is required for tracheaesophageal separation and lung lobation

doi: 10.1016/j.devcel.2019.11.002

Figure Lengend Snippet: Key Resources Table

Article Snippet: Plasmid: pCMV-Isl1 , OriGene , Cat#MR227038.

Techniques: Recombinant, Reporter Assay, Mutagenesis, Isolation, Microarray, Plasmid Preparation, Software

Journal: Nature communications

Article Title: High-efficiency motor neuron differentiation from human pluripotent stem cells and the function of Islet-1.

doi: 10.1038/ncomms4449

Figure Lengend Snippet: Figure 5 | ISL1 is a critical regulator of human motoneuron development. (a) Immunofluorescence of ISL1 (red) in cells after 15-day differentiation from H1 hESCs. Cell nuclei were stained with DAPI (4’,6-diamidino-2-phenylindole; blue). RA-D1, RA-D3 and RA-D6 denote the time of initiation of RA patterning (at day 1, day 3 and day 6, respectively). (b) Percentage of ISL1-positive cells under conditions described in a. Each bar represents mean±s.d. (error bars) of six experiments. (c) Fluorescence images of ISL1 (left), ISL1/DAPI (middle) and ISL1/ TUJ1 (right) staining in differentiated cells replated at a low density for 3 days after 15-day differentiation. (d) Fluorescence images of differentiated hESCs with NT shRNA (‘Control’, left) or with ISL1 shRNA-1 (‘ISL shRNA-1’, right) stained with antibodies against ISL1 (red) and TUJ1 (green) after 15-day differentiation (with patterning initiated at day 3). Cell nuclei were stained with DAPI (blue). (e) Fluorescence images of differentiated hESCs with NTshRNA (‘Control’, left), ISL1 shRNA-2 (‘ISL shRNA-2’, middle) and ISL1 shRNA-2 with ectopic ISL1 expression (‘shRNA-2 þ ISL1’, right) stained with antibodies against ISL1 (red) and TUJ1 (green) after 15-day differentiation (with patterning initiated at day 3). Cell nuclei were stained with DAPI (blue). (f) Fluorescence images of differentiated hESCs with NTshRNA (left) or with ISL1 shRNA (right) stained with antibodies against HB9 (red) and ChAT (green) after 20-day differentiation. Cell nuclei were stained with DAPI (blue). (g) Fluorescence images of differentiated hESCs with NT shRNA (left), ISL1 shRNA-2 (middle) and ISL1 shRNA-2, with ectopic ISL1 expression (right) stained with antibodies against HB9 (red) and ChAT (green) after 20-day differentiation (with patterning initiated at day 3). Cell nuclei were stained with DAPI (blue). All scale bars, 50 mm.

Article Snippet: For ISL1 rescue experiments, ISL1 expression vector was generated by inserting human ISL1 cDNA (OriGene SC125257) into pSin-EF2-alpha-Puro lentivector (Addgene).

Techniques: Staining, Fluorescence, shRNA, Control, Expressing

Journal: Nature communications

Article Title: High-efficiency motor neuron differentiation from human pluripotent stem cells and the function of Islet-1.

doi: 10.1038/ncomms4449

Figure Lengend Snippet: Figure 6 | Summary of the MN differentiation model and the stages of neural differentiation. (a) hESCs undergoing neural induction begin to express high levels of NPC markers, including PAX6 and SOX1 at day 3, while markedly downregulating the pluripotency markers such as OCT-4 and NANOG (albeit retaining some degree of expression). Thus, the cells at day 3 might represent a population of primitive NPCs that can be induced to become anterior NPCs and also possesses the potential to differentiate posteriorly. With neural patterning initiated at day 3 after neural induction, the early MN marker ISL1 can be seen at day 13 after hESC differentiation, while mature MN markers including HB9 and ChAT are expressed after 17-day differentiation. (b) Our results point to a previously unidentified primitive stage of neural progenitors that is intermediate between pluripotent hESCs and anterior NPCs. This population of potential primitive NPCs can be induced to become anterior NPCs in the absence of patterning factors, and also possesses the potential to differentiate along the posterior fate in the presence of patterning factors.

Article Snippet: For ISL1 rescue experiments, ISL1 expression vector was generated by inserting human ISL1 cDNA (OriGene SC125257) into pSin-EF2-alpha-Puro lentivector (Addgene).

Techniques: Expressing, Marker

Journal: Developmental cell

Article Title: Isl1 regulation of Nkx2.1 in the early foregut epithelium is required for tracheaesophageal separation and lung lobation

doi: 10.1016/j.devcel.2019.11.002

Figure Lengend Snippet: Key Resources Table

Article Snippet: The pCMV-Isl1 plasmid was purchased from OriGene.

Techniques: Recombinant, Reporter Assay, Mutagenesis, Isolation, Microarray, Plasmid Preparation, Software

Journal: Stem Cells International

Article Title: Dexamethasone Provides Effective Immunosuppression for Improved Survival of Retinal Organoids after Epiretinal Transplantation

doi: 10.1155/2019/7148032

Figure Lengend Snippet: Primers.

Article Snippet: Antibodies against the following markers were used as primary antibodies for immunofluorescence microscopy at the dilutions indicated in parentheses: Ki67 (1 : 100, A11390, ABclonal, China), CHX10 (1 : 200, AB9016, Millipore, Germany), Brn3 (1 : 200, SC-6026X, Santa Cruz, USA), Islet1 (1 : 20, BM44446, Boster, China), HuD (1 : 100, SC-48421, Santa Cruz), RBPMS (1 : 100, ab152101, Abcam), β III-tubulin (1 : 200, ab7751, Abcam), neurofilament light polypeptide (NEFL; 1 : 100, A0257, ABclonal), microtubule-associated protein 2 (MAP2, 1 : 100, BM1243, Boster), vimentin (1 : 50, BM4029, Boster), glial fibrillary acidic protein (GFAP; 1 : 500, 3670S, Cell Signaling Technology, USA), CD68 (1 : 200, ab201340, Abcam), Iba1 (1 : 200, ab5076, Abcam), and SC121 (1 : 500, Y40410, Takara Bio).

Techniques:

Journal: Stem Cells International

Article Title: Dexamethasone Provides Effective Immunosuppression for Improved Survival of Retinal Organoids after Epiretinal Transplantation

doi: 10.1155/2019/7148032

Figure Lengend Snippet: Characteristics of retinal organoids after 30-45 days of induction. The frozen sections of retinal organoids were identified by immunofluorescence staining. (a, b) At this time, most cells of the organoids were differentiating towards RGCs; they were Islet1-, Brn3- and HuD-positive. (c) But some cells outside were still retinal progenitors; they were CHX10-positive. (d, e) And in suspension cultivation, little axons were developed.

Article Snippet: Antibodies against the following markers were used as primary antibodies for immunofluorescence microscopy at the dilutions indicated in parentheses: Ki67 (1 : 100, A11390, ABclonal, China), CHX10 (1 : 200, AB9016, Millipore, Germany), Brn3 (1 : 200, SC-6026X, Santa Cruz, USA), Islet1 (1 : 20, BM44446, Boster, China), HuD (1 : 100, SC-48421, Santa Cruz), RBPMS (1 : 100, ab152101, Abcam), β III-tubulin (1 : 200, ab7751, Abcam), neurofilament light polypeptide (NEFL; 1 : 100, A0257, ABclonal), microtubule-associated protein 2 (MAP2, 1 : 100, BM1243, Boster), vimentin (1 : 50, BM4029, Boster), glial fibrillary acidic protein (GFAP; 1 : 500, 3670S, Cell Signaling Technology, USA), CD68 (1 : 200, ab201340, Abcam), Iba1 (1 : 200, ab5076, Abcam), and SC121 (1 : 500, Y40410, Takara Bio).

Techniques: Immunofluorescence, Staining, Suspension

Journal: Stem Cells International

Article Title: Dexamethasone Provides Effective Immunosuppression for Improved Survival of Retinal Organoids after Epiretinal Transplantation

doi: 10.1155/2019/7148032

Figure Lengend Snippet: Comparisons of different marker expression profiles among the control, rapamycin- (RAP-) treated, and dexamethasone- (DEX-) treated groups. Two weeks after immunosuppressant treatment, (a) cells in the retinal organoids treated with RAP expressed higher levels of the retinal ganglion cell (RGC) markers Atoh7, Islet1, and Brn3b, the dendrite marker MAP2, the retinal progenitor marker PAX6 and CHX10, and photoreceptor marker CRX, compared to levels in the control group. These cells also expressed lower levels of the axon markers TUBB3 and NEFL than cells in the control group. In DEX-treatment group cells, dendrite, retinal progenitor, and photoreceptor markers (MAP2, CHX10, and CRX) were upregulated compared to levels in the control group. (b) However, 4 weeks after immunosuppressant treatment, both RAP- and DEX-treated cells showed higher expression levels of only PAX6 and CRX as compared to those in the control group. (c) Representative histogram of flow cytometry results. Each wave represented one group; the proportion of each group was calculated by the Blk group. There were 79.87 ± 5.92% of the RGCs in the control group, 60.97 ± 7.36% in the RAP group, and 63.6 ± 10.35% in the DEX group. CTRL: control group; RAP: rapamycin-treatment group; DEX: dexamethasone-treatment group; iPSCs: negative control group in RT-PCR; Blk: blank control group in flow cytometry.

Article Snippet: Antibodies against the following markers were used as primary antibodies for immunofluorescence microscopy at the dilutions indicated in parentheses: Ki67 (1 : 100, A11390, ABclonal, China), CHX10 (1 : 200, AB9016, Millipore, Germany), Brn3 (1 : 200, SC-6026X, Santa Cruz, USA), Islet1 (1 : 20, BM44446, Boster, China), HuD (1 : 100, SC-48421, Santa Cruz), RBPMS (1 : 100, ab152101, Abcam), β III-tubulin (1 : 200, ab7751, Abcam), neurofilament light polypeptide (NEFL; 1 : 100, A0257, ABclonal), microtubule-associated protein 2 (MAP2, 1 : 100, BM1243, Boster), vimentin (1 : 50, BM4029, Boster), glial fibrillary acidic protein (GFAP; 1 : 500, 3670S, Cell Signaling Technology, USA), CD68 (1 : 200, ab201340, Abcam), Iba1 (1 : 200, ab5076, Abcam), and SC121 (1 : 500, Y40410, Takara Bio).

Techniques: Marker, Expressing, Control, Flow Cytometry, Negative Control, Reverse Transcription Polymerase Chain Reaction

Journal: Neuron

Article Title: Sox11 Expression Promotes Regeneration of Some Retinal Ganglion Cell Types but Kills Others

doi: 10.1016/j.neuron.2017.05.035

Figure Lengend Snippet: (A, B) Representative images of optic nerve sections showing CTB-labeled axons in wild type mice with intravitreal injections of AAV expressing PLAP, Sox4, and Sox11 (A), and Pax6, Math5, Sox2, Brn3b, and Isl1 (B) at 2 weeks after optic nerve injury. The crush site is indicated with a red asterisk. Scale bars in (A) and (B) represent 250 µm.

Article Snippet: In vivo procedures and reagents Production of AAVs Vectors of AAV-Pax6 and AAV-Isl1 vectors were made by inserting mouse Pax6 cDNA (Addgene #32932) and mouse Isl1 cDNA (Addgene #32929) respectively into an AAV plasmid consisting of the cytomegalovirus enhancer fused to the chicken beta-actin promoter (CAG promoter) and Woodchuck hepatitis virus Posttranscriptional Regulatory Element (WPRE).

Techniques: Labeling, Expressing