Journal: Nature

Article Title: Intratumoral heterogeneity generated by Notch signaling promotes small cell lung cancer

doi: 10.1038/nature22323

Figure Lengend Snippet: a , Quantification of the frequency (Freq.) of Hes1 pos cells in TKO tumors at different stages of tumor development. Scores: 0 (0%), 1 (1-20%), 2 (20-60%), 3 (>60%). Hyperplasias ( n =26, from 5 mice, defined by area < 16,600 μm 2 ) and early tumors ( n =69, from 5 mice) were analyzed 3 months after Ad-CMV-Cre); late tumors ( n =83, from 5 mice) and liver metastases ( n =54, from 5 mice) were analyzed 6-7 months after Ad-CMV-Cre (data for the late tumors are shown for comparison and are the same as in ). b , As in a, for human SCLC tumor microarray sections and segregated by clinical stage. H-scores: stage I (123.9; n =71 sections), stage II (135.3; n =68 sections), stages III+IV (148.5; n =33 sections). c , Representative immunofluorescence (IF) for GFP in TKO Hes1 GFP/+ SCLC tumors. d , Representative flow cytometry plots of cells isolated from pooled tumors from a TKO Hes1 GFP/+ mouse. Arrows depict the sequential gating strategy for enriching for single, live (by exclusion of 7-aminoactinomycin D (7-AAD)) and lineage (CD45, CD31, TER-119)-negative cells. CD24 labels >98% of Cre-recombined cells and thus further enriches for tumor cells . e , IF of a TKO Rosa26 lox-stop-lox-tdTomato ;Hes1 GFP/+ tumor section showing co-localization of GFP and Tomato signals. f , Flow cytometry shows that GFP high cells in pooled tumors from a TKO Rosa26 lox-stop-lox-tdTomato ;Hes1 GFP/+ mouse infected with Ad-CMV-Cre are positive for Tomato expression (representative of n =2 mice). g , Genotyping PCR analysis for recombination (Δ) or the unrecombined (floxed) alleles at the Rb , p53 and p130 loci in GFP neg and GFP high tumor cells sorted from 3 TKO Hes1 GFP/+ mice. DNA from fl/fl and/or Δ/Δ cells serve as controls. Scale bars, 50 μm.

Article Snippet: TKO Hes1 GFP/+ mice were injected intraperitoneally with 100mg/kg EdU (5-ethynyl-2’-deoxyuridine; Life Technologies) 8 hours before euthanasia.

Techniques: Comparison, Microarray, Immunofluorescence, Flow Cytometry, Isolation, Infection, Expressing

Journal: Nature

Article Title: Intratumoral heterogeneity generated by Notch signaling promotes small cell lung cancer

doi: 10.1038/nature22323

Figure Lengend Snippet: a , Schematic for the generation of TKO allografts. A TKO tumor was isolated, digested to form single cells and implanted subcutaneously in NSG mice. b , Representative hematoxylin and eosin (H&E) staining and IHC for Ascl1 in allograft tumor sections. c , Tumor volumes of TKO allografts treated with the indicated drugs ( n =16 tumors per group, 1 experiment). The experiment was stopped at day 18 and samples were collected for analysis. d,e , Representative IHC for Hes1 (d) or double IHC for Hes1 (brown) and Ascl1 (pink) (e) in TKO allograft tumors collected 18 days after the start of treatment. f,g , IHC staining quantification for the frequency of Hes1 pos CC3 pos (f) and Hes1 pos Ki67 pos (g) cells in murine TKO allografts after 18 days of treatment ( n =8 tumors per group, except for C/I and tarex in (f) ( n =7 tumors each). h,i , IHC staining quantification for the frequency of HES1 pos CC3 pos (h) and HES1 pos KI67 pos (i) cells in xenograft tumors after 31 days of treatment (for h, control/tarex groups: n =4 tumors each; C/I: n =3 tumors; tarex+C/I: n =2 tumors; for i, control/tarex groups: n =4 tumors each; C/I and tarex+C/I groups: n =5 tumors each). j,k , IHC staining quantification for the frequency of Ascl1 pos CC3 pos (j) and Ascl1 pos Ki67 pos (k) cells in murine TKO allografts after 18 days of treatment ( n =8 tumors per group, except for the C/I group in (j) ( n =7 tumors). l,m , IHC staining quantification for the frequency (%) of ASCL1 pos CC3 pos (l) and ASCL1 pos KI67 pos (m) cells in xenograft tumors after 31 days of treatment ( n =4 tumors per group, except for control and tarex groups in (l) ( n =2 tumors) and the tarex+C/I group in (m) ( n =3 tumors). n,o , Representative IHC (n) and quantification of HES1 pos (o) cells in LU66 PDX tumors collected 56 days after the start of treatment (control: n =3 tumors; tarex: n =4 tumors; C/I and tarex+C/I: n =5 tumors per group). *P < 0.05; **P < 0.01; ***P < 0.001; **** P < 0.0001. Statistical significance was determined by two-tailed unpaired Student’s t -test. Data are represented as mean ± SEM. Scale bars, 50 μm.

Article Snippet: TKO Hes1 GFP/+ mice were injected intraperitoneally with 100mg/kg EdU (5-ethynyl-2’-deoxyuridine; Life Technologies) 8 hours before euthanasia.

Techniques: Isolation, Staining, Immunohistochemistry, Control, Two Tailed Test

Journal: Nature

Article Title: Intratumoral heterogeneity generated by Notch signaling promotes small cell lung cancer

doi: 10.1038/nature22323

Figure Lengend Snippet: a,b , Representative Hes1 IHC (a) and frequency of Hes1 pos cells (b) in mouse SCLC ( n =5 mice, 83 tumors). c,d , As in a,b, for human SCLC ( n =172 sections). e , Flow cytometry of %GFP high cells from pooled TKO Hes1 GFP/+ tumors ( n =18 mice). f , qRT-PCR of Notch pathway genes in GFP high relative to GFP neg tumor cells ( n =3 mice). g , As in e, with mice treated with DMSO ( n =5 mice) or DBZ ( n =4 mice). h , Immunoblots of GFP high cell lines grown with Dll4 or 72 hours after removal from Dll4. i , GFP and Uchl1 (NE marker) immunofluorescence in TKO Hes1 GFP/+ tumors (representative of n =3 biological replicas). j , Images of cell cultures established from GFP neg and GFP high tumor cells (representative of n =5 biological replicas). Inset: NE KP1 SCLC cell line. k , EdU incorporation in cells sorted from TKO Hes1 GFP/+ tumors ( n =3 mice). *P < 0.05; **P < 0.01; ***P < 0.001. Two-tailed paired (f,k) or unpaired (g) Student’s t -test. Data are mean ± s.d. Scale bars, 50 μm.

Article Snippet: TKO Hes1 GFP/+ mice were injected intraperitoneally with 100mg/kg EdU (5-ethynyl-2’-deoxyuridine; Life Technologies) 8 hours before euthanasia.

Techniques: Flow Cytometry, Quantitative RT-PCR, Western Blot, Marker, Immunofluorescence, Two Tailed Test

Journal: Nature

Article Title: Intratumoral heterogeneity generated by Notch signaling promotes small cell lung cancer

doi: 10.1038/nature22323

Figure Lengend Snippet: a , Unsupervised clustering of qRT-PCR ( n =46 cells) from one TKO Hes1 GFP/+ tumor (representative of n =2 experiments). Dark blue: undetectable expression. Arrowheads: NE cells that express at least one Notch receptor but have undetectable Hes1 . b , Flow cytometry of freshly isolated GFP neg cells grown on Dll4 or PBS control (representative of n =3 biological replicas). c , Immunoblot of (b). GFP neg cells grown on Dll4 were re-sorted for GFP neg and GFP high populations. d , Relative number of GFP high cells generated from freshly-isolated GFP neg cells grown on Dll4 after DBZ treatment ( n =3 biological replicas). e , Flow cytometry of a GFP high cell line treated with DMSO or DBZ while grown with or without Dll4 or co-cultured with 3 NE cell lines without Dll4 ( n =3 biological replicas). Median GFP intensity normalized to DMSO; -Dll4. f , Freshly isolated GFP neg cells that became GFP high after Dll4 exposure were replated with or without Dll4. Flow cytometry and images are shown (representative of n =2 biological replicas). *P < 0.05; **P < 0.01; ***P < 0.001. Two-tailed paired Student’s t -test. Data are mean ± s.d. Scale bars, 50 μm.

Article Snippet: TKO Hes1 GFP/+ mice were injected intraperitoneally with 100mg/kg EdU (5-ethynyl-2’-deoxyuridine; Life Technologies) 8 hours before euthanasia.

Techniques: Quantitative RT-PCR, Expressing, Flow Cytometry, Isolation, Control, Western Blot, Generated, Cell Culture, Two Tailed Test

Journal: Nature

Article Title: Intratumoral heterogeneity generated by Notch signaling promotes small cell lung cancer

doi: 10.1038/nature22323

Figure Lengend Snippet: a , Cell viability assay of GFP neg , GFP high or a mixture of GFP neg and GFP high cells (bulk) in Matrigel ( n =3 biological replicas). b,c , Relative luciferase activity (b) and EdU incorporation (c) of luciferase-labeled KP1 cells in 2% serum with or without GFP high cells ( n =3 biological replicas, with n =3 technical replicas each for (b)). d,e , As in b,c, with conditioned media from KP1 or from GFP high cell lines ( n =3 biological replicas, with n =3 technical replicas each for (d)). f , Quantification of %CC3 pos cells in GFP neg or GFP high tumor cells in TKO Hes1 GFP/+ mice treated acutely with cisplatin/etoposide ( n =15 tumors per group, from 3 mice). g , Tumor volumes of TKO SCLC allografts ( n =5 tumors per group, one experiment). h , Quantification of Hes1 pos cells ( n =8 tumors except tarex+C/I ( n =6 tumors), one experiment) in TKO allografts after 18 days of treatment. i , Tumor volumes of PDX model OMP-LU66 treated with control antibody ( n =10 tumors), tarex ( n =9), C/I ( n =20), or tarex + C/I ( n =24) (one experiment). j , Model. In SCLC tumors, Notch signaling is activated in a subset of NE cells, leading to a loss of NE features. Non-NE SCLC cells promote the growth of NE cells. Combining chemotherapy and Notch inhibition can target both the fast cycling NE cells and the non-NE cells, respectively. *P < 0.05; **P < 0.01; *** P < 0.001; **** P < 0.0001. For a-f, statistical significance determined by two-tailed paired (a-e) or unpaired (f) Student’s t -test; data are mean ± s.d. For g-i, statistical significance determined by two-tailed unpaired t-test against the control group when applicable or between the C/I and Tarex+C/I groups; data are mean ± SEM. Scale bars, 50 μm.

Article Snippet: TKO Hes1 GFP/+ mice were injected intraperitoneally with 100mg/kg EdU (5-ethynyl-2’-deoxyuridine; Life Technologies) 8 hours before euthanasia.

Techniques: Viability Assay, Luciferase, Activity Assay, Labeling, Control, Inhibition, Two Tailed Test

Journal: Nature

Article Title: Intratumoral heterogeneity generated by Notch signaling promotes small cell lung cancer

doi: 10.1038/nature22323

Figure Lengend Snippet: a,b , Co-immunofluorescence for GFP and EdU (a) or phospho-histone H3 (b) in TKO Hes1 GFP/+ tumors. c , Weight of tumors formed from freshly sorted GFP neg and GFP high tumor cells implanted subcutaneously in immunocompromised NSG mice ( n =6 tumors each; two-tailed unpaired Student’s t -test). d , Flow cytometry of GFP in a tumor formed from freshly sorted GFP neg cells implanted subcutaneously in immunocompromised NSG mice (representative of n =6 biological replicas). *P < 0.05. Data are represented as mean ± s.d. Scale bars, 50 μm.

Article Snippet: TKO Hes1 GFP/+ mice were injected intraperitoneally with 100mg/kg EdU (5-ethynyl-2’-deoxyuridine; Life Technologies) 8 hours before euthanasia.

Techniques: Immunofluorescence, Two Tailed Test, Flow Cytometry

Journal: Nature

Article Title: Intratumoral heterogeneity generated by Notch signaling promotes small cell lung cancer

doi: 10.1038/nature22323

Figure Lengend Snippet: a , Representative IHC for Hes1 and cleaved caspase-3 (CC3) in serial TKO tumor sections. Inset: higher magnification of a positive control for CC3 (tumor from a mouse treated with chemotherapy). b , Predicted numbers and ratio of GFP neg and GFP high tumor cells if the two populations divide independently of each other, and GFP neg cells cycle approximately 3 times faster than GFP high cells. c , Representative IHC in serial sections from TKO Hes1 GFP/+ tumors initiated by Adeno-CGRP-Cre. d , Quantification of the frequency (Freq.) of Hes1 pos cells in TKO hyperplasias ( n =23, from 5 mice) and tumors ( n =50, from 7 mice) induced by Adeno-CGRP-Cre. Scores: 0 (0%), 1 (1-20%), 2 (20-60%) 3 (>60%). e , Flow cytometry of %GFP high cells in pooled tumors from TKO Hes1 GFP/+ mice ( n =4) infected with Ad-CGRP-Cre. f , Images of freshly isolated GFP neg cells grown on dishes coated with Dll4 or PBS control (representative of n =3 biological replicas). g,h , Freshly isolated GFP neg cells that remained GFP neg after culture on Dll4-coated dishes were replated on dishes coated with Dll4 ligand (+Dll4) or PBS control (-Dll4). g , Flow cytometry and images (representative of n =2 biological replicas). h , GFP neg and GFP high cells that formed after this second round of Dll4 stimulation were sorted and analyzed by immunoblot. Control: GFP high cell line. i , Relative number of GFP high cells formed from freshly isolated GFP neg cells grown on Dll4 after two weeks of tarextumab treatment ( n =3 biological replicas each). j , Single cell qRT-PCR ( n =45 each) of H29, H82 and H889 human SCLC cell lines. Heatmap was generated by unsupervised clustering of each cell line. Dark blue regions indicate undetectable expression. k , qRT-PCR for HES1 after 72 hours of culture with or without Dll4. Data is normalized to GAPDH ( n =3 biological replicas with n =3 technical replicas each). l,m , GFP high cell lines were treated with DMSO or DBZ while grown with or without Dll4 or co-cultured with 3 individual NE cell lines in the absence of Dll4. GFP expression was analyzed by flow cytometry after 72 hours. l , Flow cytometry of GFP high cell line #1 (representative of n =3 biological replicas; GFP intensity quantified in ). m , Quantification of GFP intensity in GFP high cell line #2; relative median GFP intensity normalized to the DMSO -Dll4 condition ( n =3 biological replicas). n,o , Representative images (n) and qRT-PCR (o) of GFP high cell lines cultured in the presence of absence of Dll4 for more than a month ( n =3 biological replicas with n =3 technical replicas). p , Freshly isolated GFP neg cells that became GFP high after culture on Dll4 were replated on dishes coated with Dll4 ligand or PBS control and analyzed by immunoblot after a month. GFP neg cell lysate: positive control for Ascl1. *P < 0.05; **P < 0.01. Statistical significance was determined by two-tailed paired Student’s t -test. Data are represented as mean ± s.d. Scale bars, 50 μm.

Article Snippet: TKO Hes1 GFP/+ mice were injected intraperitoneally with 100mg/kg EdU (5-ethynyl-2’-deoxyuridine; Life Technologies) 8 hours before euthanasia.

Techniques: Positive Control, Flow Cytometry, Infection, Isolation, Control, Western Blot, Quantitative RT-PCR, Generated, Expressing, Cell Culture, Two Tailed Test

Journal: Nature

Article Title: Intratumoral heterogeneity generated by Notch signaling promotes small cell lung cancer

doi: 10.1038/nature22323

Figure Lengend Snippet: a , Flow cytometry showing gates used to sort for tumor cells with negative (neg), intermediate (int), and high levels of GFP from a TKO Hes1 GFP/+ mouse (representative of n =4 biological replicas). b , qRT-PCR for Notch pathway genes and NE genes in sorted cells ( n =4 biological replicas with n =3 technical replicas each). c-e , Single cell qRT-PCR of GFP neg , GFP int and GFP high cells sorted from one TKO Hes1 GFP/+ tumor ( n =32 cells sorted per group; 20 cells with low expression of housekeeping genes were excluded from the heatmaps). (c) Unsupervised clustering segregates the cells into two main groups. (d) Supervised clustering and (e) analysis of Hes1 mRNA (normalized to Gapdh ) shows that the number of GFP int cells with detectable Hes1 levels is intermediate between GFP neg and GFP high cells. f , Representative images of freshly isolated cells in culture (without Dll4). Scale bars, 50 μm. *P < 0.05; **P < 0.01; ***P < 0.001; **** P < 0.0001. Statistical significance was determined by two-tailed paired Student’s t -test. Data are represented as mean ± s.d.

Article Snippet: TKO Hes1 GFP/+ mice were injected intraperitoneally with 100mg/kg EdU (5-ethynyl-2’-deoxyuridine; Life Technologies) 8 hours before euthanasia.

Techniques: Flow Cytometry, Quantitative RT-PCR, Expressing, Isolation, Two Tailed Test

Journal: Nature

Article Title: Intratumoral heterogeneity generated by Notch signaling promotes small cell lung cancer

doi: 10.1038/nature22323

Figure Lengend Snippet: a , GSEA signatures enriched in GFP high (astroglial signature) and GFP neg (neuronal signature) tumor cells based on microarray data of tumors from n =3 mice. b , Images of GFP neg , GFP high or bulk tumor cells (mixture of GFP neg and GFP high ) 7 days after isolation from TKO Hes1 GFP/+ mice and seeded at equal numbers in 50% Matrigel (representative of n =3 biological replicas). c , Immunofluorescence for Uchl1 (NE marker) and GFP 10 days after the cells were sorted from TKO Hes1 GFP/+ tumors and in 50% Matrigel. Note: Most of the spheroids in the bulk culture were composed of only one type of cell (Uchl1 pos GFP neg or Uchl1 neg GFP pos ; the mixed spheroid shown here are the minority. d , Quantification of the number of each type of spheroids in each culture condition from (c) ( n =3 mice, 2 sections each for immunostaining). e-g , Relative luciferase activity of a luciferase-labeled mouse SCLC NE cell line seeded alone or co-cultured with GFP high cell lines ( n =2 (e) or n =3 (f,g) biological replicas with n =3 technical replicas each). e , 2% serum, KP2 NE cell line. f,g , 10% serum, KP1 (f) and KP2 (g) cell lines. h-j , As in e-g, but the cells were fixed and collected for EdU analysis by flow cytometry ( n =2 (h) or n =3 (i,j) biological replicas). k , Representative images of KP1 cells seeded in conditioned media (CM) (2% serum) from KP1 cells or from GFP high cell lines. l , Relative luciferase activity of luciferase-labeled KP2 cells 72 hours after seeding in CM (2% serum) from KP2 cells or from GFP high cell lines ( n =3 biological replicas with n =3 technical replicas each). m , As in l, but the cells were fixed and collected for EdU analysis by flow cytometry ( n =3 biological replicas). n,o , As in l, but with KP1 (n) or KP2 cells (o) in 10% serum ( n =3 biological replicas with n =3 technical replicas each). p,q , AlamarBlue cell viability assay for NE SCLC cell lines 72 hours after culture with the indicated recombinant proteins ( n =3 biological replicas with n =3 technical replicas each). r , ELISA assay for midkine in supernatant from NE ( n =4) and GFP high ( n =4) cell lines ( n =2 biological replicas each). s , Luminex assay for midkine in serum plasma from normal (control; n =11) and SCLC ( n =15) patients. t , Relative luciferase activity of luciferase-labeled mouse SCLC NE cell lines ( n =4) 72 hours after seeding alone or co-cultured with GFP high cell lines and treated with 10 μM DBZ or DMSO control. Data are normalized to NE-monoculture with DMSO ( n =3 biological replicas with n =3 technical replicas each). *P < 0.05; **P < 0.01; ***P < 0.001. Statistical significance was determined by two-tailed paired Student’s t -test, except for (r,s: two-tailed unpaired Student’s t -test). Data are represented as mean ± s.d. Scale bars, 50 μm.

Article Snippet: TKO Hes1 GFP/+ mice were injected intraperitoneally with 100mg/kg EdU (5-ethynyl-2’-deoxyuridine; Life Technologies) 8 hours before euthanasia.

Techniques: Microarray, Isolation, Immunofluorescence, Marker, Immunostaining, Luciferase, Activity Assay, Labeling, Cell Culture, Flow Cytometry, Viability Assay, Recombinant, Enzyme-linked Immunosorbent Assay, Luminex, Clinical Proteomics, Control, Two Tailed Test

Journal: Nature

Article Title: Intratumoral heterogeneity generated by Notch signaling promotes small cell lung cancer

doi: 10.1038/nature22323

Figure Lengend Snippet: a,b , Average cell viability (MTT assay) of NE ( n =4) and GFP high ( n =3) cell lines 48 hours after cisplatin (a) or etoposide (b) treatment ( n =3 biological replicas with n =3 technical replicas each). c,d , Quantification of cells expressing cleaved caspase-3 (CC3) (c) and Hes1 (d) in tumors in TKO mice acutely treated with cisplatin and etoposide (CC3: n =49; Hes1: n =41 tumors) or a vehicle control (CC3: n =48; Hes1: n =67 tumors) (data from 2 groups of 3 mice previously described in in but analyzed as number of CC3 pos per tumor, not per mouse). e , Representative immunofluorescence of tumors from TKO Hes1 GFP/+ mice acutely treated with acutely treated with cisplatin and etoposide. Scale bars, 50 μm. f , Quantification of Hes1 pos cells in the tumors of TKO Rosa26 lox-stop-lox-Luciferase mice after 3 weeks of weekly saline ( n =190 tumors, from 7 mice) or cisplatin ( n =217 tumors, from 8 mice) treatments. g-i , Overall survival of all ( g ; n =65, 49 HES1 neg , 16 HES1 pos ), stages I-III ( h ; n =26, 19 HES1 neg , 7 HES1 pos ) or stage IV ( i ; n =39, 30 HES1 neg , 9 HES1 pos ) SCLC patients with HES1-negative or HES1-positive tumors. j-l , Progression-free survival of all ( j ; n =67, 51 HES1 neg , 16 HES1 pos ), stages I-III ( k ; n =28, 21 HES1 neg , 7 HES1 pos ) or stage IV ( l ; n =39, 30 HES1 neg , 9 HES1 pos ) SCLC patients with HES1-negative or HES1-positive tumors. *P < 0.05; **P < 0.01; ***P < 0.001; **** P < 0.0001. Statistical significance was determined by two-tailed paired (a,b) or unpaired (c,d,f) Student’s t -test. For g-l, probability was calculated using a weighted log-rank test with emphasis on later time points (see methods). Data are represented as mean ± s.d.

Article Snippet: TKO Hes1 GFP/+ mice were injected intraperitoneally with 100mg/kg EdU (5-ethynyl-2’-deoxyuridine; Life Technologies) 8 hours before euthanasia.

Techniques: MTT Assay, Expressing, Control, Immunofluorescence, Luciferase, Saline, Two Tailed Test

Journal: Frontiers in Neuroscience

Article Title: Co-grafts of Human Embryonic Stem Cell Derived Retina Organoids and Retinal Pigment Epithelium for Retinal Reconstruction in Immunodeficient Retinal Degenerate Royal College of Surgeons Rats

doi: 10.3389/fnins.2021.752958

Figure Lengend Snippet: Generation of hESC derived retina organoids (CRX-GFP cell line) and RPE (H9 cell line). (A) Phase contrast images of organoid development up to day 56. (B–D) Progenitor gene expression analysis of CRX-GFP hESC-derived retina organoids ( n = 2) and undifferentiated stem cells ( n = 1). (B) OCT4 expression is absent in differentiated day 48 retina organoids. (C) Retina organoids (red) highly express genes indicative of primitive retina and photoreceptor progenitors. (D) Markers for mature, terminally differentiated retinal subpopulations. Retina organoids (red) contain markers for all the major retinal cell types, including photoreceptor progenitors, glia, ganglion cells, bipolar, horizontal, amacrine, and Müller cells. (E) Flow analysis shows that GFP expression is absent in the undifferentiated stem cells, early embryoid bodies, and young retina organoids. A distinct GFP+ population appears at day 70. Expression is highest day 78 and persists in the oldest analyzed day 91 organoids. (F) Examples of gates drawn used for FACS sorting of the GFP negative and GFP positive populations. (G) Post-FACS purities >90%. Cells were gated based on singlets, SSC vs. FSC profile, and if they were DAPI negative. Negative controls were undifferentiated CRX-GFP hESC (day 0). (H) Parylene only implant (no cells). (I) CPCB-RPE1 implant covered with hESC-RPE cells. (J) Enlargement of (I) to show details of hESC-RPE cells forming an epithelium (28–35 days culture).

Article Snippet: The first one, the cone-rod homeobox gene-green fluorescent protein (CRX-GFP) hESC line , was maintained with TeSR E8 media on Vitronectin XF (Stemcell Technologies) coated plates.

Techniques: Derivative Assay, Gene Expression, Expressing

Journal: Frontiers in Neuroscience

Article Title: Co-grafts of Human Embryonic Stem Cell Derived Retina Organoids and Retinal Pigment Epithelium for Retinal Reconstruction in Immunodeficient Retinal Degenerate Royal College of Surgeons Rats

doi: 10.3389/fnins.2021.752958

Figure Lengend Snippet: Transplant histology at 2 months (A,B) and 6.5 months (C–E) post-surgery. (A,C) Co-graft area. (B,D) Host retina outside co-graft. (A–D) H&E staining. White asterisks indicate the center of rosettes (spherical arrangement of photoreceptors and other retinal layers around a central lumen). The transplant in (C) has grown around the parylene RPE implant (transplant #3 in , 197 dps). (E) Bestrophin (gold) and DAPI (blue) shows RPE and parylene in transplant (#2 in , 195 dps). Blue arrows point to parylene. Red arrows point to hESC-RPE. Yellow arrows point to host RPE. The separation of tissue is processing artifact. Scale bars = 100 μm (A–D) ; 20 μm (E) .

Article Snippet: The first one, the cone-rod homeobox gene-green fluorescent protein (CRX-GFP) hESC line , was maintained with TeSR E8 media on Vitronectin XF (Stemcell Technologies) coated plates.

Techniques: Staining

Journal: Current protocols in human genetics

Article Title: Differentiation and Contractile Analysis of GFP-Sarcomere Reporter hiPSC-Cardiomyocytes

doi: 10.1002/cphg.53

Figure Lengend Snippet: Protocol for hiPSC-CM differentiation of genome-edited, GFP-sarcomere fusion hiPSCs. This small-molecule mediated differentiation approach involves modulation of the Wnt cell signaling pathway to induce cardiac mesoderm and cardiomyocytes. The earliest GFP-sarcomere signal will coincide with the earliest sarcomeric protein expression and will strengthen over the course of differentiation.

Article Snippet: Materials list-behavior=simple prefix-word= mark-type=none max-label-size=0 Genome-edited, GFP-sarcomere fusion hiPSCs Matrigel (hESC-qualified; BD Sciences, cat. no. 354277) DMEM/F12 medium (Thermo Fisher Scientific, cat. no. 11320033) RPMI 1640 medium (Thermo Fisher Scientific, cat. no. 11835055) Glucose-free RPMI 1640 medium (Thermo Fisher Scientific, cat. no. 11879-020) B27 Supplement Minus Insulin (Thermo Fisher Scientific, cat. no. A1895601) B27 Supplement with Insulin (Thermo Fisher Scientific, cat. no. 17504-044) CHIR99021 (Thermo Fisher Scientific, cat. no. 508306) Wnt-C59 (Biorbyt, cat. no. orb181132) 6-well tissue culture-treated plates Standard cell culture laminar flow hood mTeSR1 medium (StemCell Technologies, cat. no. 05850) Rho kinase inhibitor (ROCKi; Tocris cat. no. 1254) 0.5M EDTA (Thermo Fisher Scientific, cat. no. 15575020) DMSO (Sigma-Aldrich, cat. no. D-2650) Differentiate Genome-Edited, GFP-Sarcomere Fusion hiPSCs to Cardiomyocytes list-behavior=enumerated prefix-word= mark-type=decimal max-label-size=0 Prepare 6-well plates coated with Matrigel extracellular matrix, on which genome-edited, fluorescent fusion reporter hiPSCs will grow in a feeder cell-free format prior to differentiation.

Techniques: Expressing