Journal: Cell reports

Article Title: Live Imaging Reveals that the First Division of Differentiating Human Embryonic Stem Cells Often Yields Asymmetric Fates.

doi: 10.1016/j.celrep.2017.09.044

Figure Lengend Snippet: Figure 2. Innate Resistance to WNT Signaling in One Daughter Cell Leads to Asymmetric Primitive Streak Specification in Daughter Cell Pairs (A) Overview of the WNT pathway. (B) Stills taken from live imaging of hESCs differentiating toward primitive streak (top); staining of fixed cells reveals asymmetric LEF1 protein expression (bottom). Scale bar represents 10 mm. (C) Ratio of LEF1 protein expression in pairs of daughter cells derived from hESCs exposed to primitive streak differentiation media (ACTIVIN, BMP4, FGF2, and WNT3A); the ratio of LEF1 expression is plotted against the number of hours since the mother cell divided prior to the end of live imaging. Pie charts enumerate the overall percentage of asymmetric versus symmetric daughter cell pairs. (D) Live imaging of hESCs carrying a Tcf/Lef-TurboGFP:NLS:d2PEST reporter allele revealed that upon treatment with primitive streak differentiation media (ACTIVIN, BMP4, FGF2, and WNT3A), WNT/b-catenin signaling is often only activated in one cell in a daughter cell pair. Scale bar represents 10 mm. (E) Ratio of Tcf/Lef-TurboGFP fluorescence in pairs of daughter cells, plotted against the number of hours since the mother cell divided prior to the end of live imaging. Pie charts enumerate the overall percentage of asymmetric versus symmetric daughter cell pairs. (F) MIXL1-GFP hESCs differentiated in primitive streak differentiation media (ACTIVIN, BMP4, FGF2) containing the GSK3 inhibitor CHIR99021 (3 mM) still differentiated through asymmetric divisions, as shown by still images from live imaging. Immunostaining of fixed cells revealed that the MIXL1-GFP+ daughter also expressed BRACHYURY protein. Scale bar represents 5 mm. (G and H) Ratio of MIXL1-GFP (G) and BRACHYURY (H) protein expression in pairs of CHIR99021-treated daughter cells, plotted against the number of hours since the mother cell divided prior to the end of live imaging. Pie charts enumerate the overall percentage of asymmetric versus symmetric divisions. Data are representative of three to five independent experiments.

Article Snippet: Atop this minimal primitive streak induction condition, WNT3A protein (made in house from WNT3A-producing L cells), RSPO2 protein (10–20 ng/mL; R&D Systems), and/or the small-molecule WNT agonist CHIR99021 (3 mM; Tocris) were added as indicated to potentiateWNT signaling and to drive primitive streak specification.

Techniques: Imaging, Staining, Expressing, Derivative Assay, Immunostaining

Journal: Nature biomedical engineering

Article Title: Generation of neural organoids for spinal-cord regeneration via the direct reprogramming of human astrocytes

doi: 10.1038/s41551-022-00963-6

Figure Lengend Snippet: a , Schematic for the direct reprogramming of human astrocytes into neurons. b , Schematic diagram of primary screening protocol to convert human astrocytes into neurons using each of 15 small molecules. AM, Astrocyte Medium; NB, Neurobasal medium. c , Representative images of MAP2 + cells treated with each of 15 small molecules and DMSO as control at day 21. Scale bar, 100 μm. d , Quantification of the percentage of induced MAP2 + neurons treated with 15 small molecules (CHIR99021, Y27632, SB431542, XAV939 and SAG, n = 4 independent experiments; the rest, n = 3 independent experiments) and DMSO (n = 4 independent experiments). Data are presented as mean ± SEM.

Article Snippet: From day 3 to day 8, small molecules including CHIR99021 (1.5 μM, Tocris), VPA (5 mM, Cayman), RepSox (1 μM, Tocris), Forskolin (10 μM, Cayman), JQ1 (50 nM, Sigma), ISX-9 (10 μM, Tocris), LDN193189 (100 nM, Stemolecule), TTNPB (0.5 μM, Tocris), Y27632 (10 μM, Tocris), DAPT (2.5 μM, Sigma), SAG (100 nM, EMD Millipore), SB431542 (10 μM, MCE), PD0325901 (10 μM, Tocris), SU5402 (10 μM, Biovision), and XAV939 (10 μM, Tocris) were added to induce neurons cultured with Neural Medium for primary screening.

Techniques: Control

Journal: Nature biomedical engineering

Article Title: Generation of neural organoids for spinal-cord regeneration via the direct reprogramming of human astrocytes

doi: 10.1038/s41551-022-00963-6

Figure Lengend Snippet: a , Representative images of MAP2 + cells treated with different combinations of small molecules and DMSO. C, CHIR99021; SB, SB431542; R, RepSox; Y, Y27632. Scale bar, 100 μm. b , Quantitative analysis of MAP2 + cells treated with small molecular combinations (CSBY, CSBRY group, n = 4 independent experiments; the rest, n = 3 independent experiments) and DMSO (n = 4 independent experiments). c , Representative images of MAP2 + neurons converted from hESCs-derived astrocytes treated by CSBRY cocktail. Scale bar, 100 μm. d , Quantitative analysis of MAP2 + neurons converted from hESCs-derived astrocytes treated by CSBRY (n = 3 independent experiments). e , Representative images of MAP2 + neurons converted from human mature astrocytes isolated from the patients with glioma treated by CSBRY combination. Scale bar, 100 μm. f , Quantitative analysis of MAP2 + neurons converted from human mature astrocytes isolated from the patients with glioma treated by CSBRY (n = 3 independent experiments). NP: gliomas patient. Data are presented as mean ± SEM. Unpaired Student’s t-test was used for comparing two groups.

Article Snippet: From day 3 to day 8, small molecules including CHIR99021 (1.5 μM, Tocris), VPA (5 mM, Cayman), RepSox (1 μM, Tocris), Forskolin (10 μM, Cayman), JQ1 (50 nM, Sigma), ISX-9 (10 μM, Tocris), LDN193189 (100 nM, Stemolecule), TTNPB (0.5 μM, Tocris), Y27632 (10 μM, Tocris), DAPT (2.5 μM, Sigma), SAG (100 nM, EMD Millipore), SB431542 (10 μM, MCE), PD0325901 (10 μM, Tocris), SU5402 (10 μM, Biovision), and XAV939 (10 μM, Tocris) were added to induce neurons cultured with Neural Medium for primary screening.

Techniques: Derivative Assay, Isolation