pd 407824  (Santa Cruz Biotechnology)

Journal: Regenerative Therapy

Article Title: Addition of Chk1 inhibitor and BMP4 cooperatively promotes retinal tissue formation in self-organizing human pluripotent stem cell differentiation culture

doi: 10.1016/j.reth.2021.12.003

Figure Lengend Snippet: Chk1i PD407824 combined with rhBMP4 treatment promotes retinal differentiation from hESCs . (A) Scheme of retinal differentiation. rhBMP4 and 1 μM PD (Chk1i) were added in the differentiation medium on day 3. The concentration of rhBMP4 and PD were diluted into half by half medium change on every 3–4 days. (B-C) Examination of three candidate small molecule BMP signal activators for induction of Rx:: Venus expression using hESCs Rx:: Venus reporter line. Bright-field view with expression of Rx:: Venus of day 9 aggregates after small molecule treatment. (B) Treatment with small molecules alone. (C) PD treatment combined with rhBMP4. Scale bar, 200 μm (B, C), SJ: SJ000286237, PD: PD407824.

Article Snippet: SB-4 (#6881) and PD407824 (#2694) were purchased from R&D Systems.

Techniques: Concentration Assay, Expressing

Journal: Regenerative Therapy

Article Title: Addition of Chk1 inhibitor and BMP4 cooperatively promotes retinal tissue formation in self-organizing human pluripotent stem cell differentiation culture

doi: 10.1016/j.reth.2021.12.003

Figure Lengend Snippet: Treatment with low-concentration rhBMP4 and Chk1i PD407824 efficiently causes retinal differentiation reducing an Rx:: Venus-negative cell clump s . (A) Bright-field view with Rx:: Venus fluorescence in hESC-derived 3D-retina on day 14. Images were obtained on an individual low cell binding 96-well V-bottomed plate (upper image) or a 90 mm Petri dish after the transfer for long-term culture (lower image). (B) Quantification analysis of 3D-retinas without an Rx:: Venus-negative cell clump. Data represent mean ± s.e.m. (n = 3 with 32 3D-retinas). Bars represent the proportion of the 3D-retina without a non-retinal cell clump. Scale bars, 500 μm.

Article Snippet: SB-4 (#6881) and PD407824 (#2694) were purchased from R&D Systems.

Techniques: Concentration Assay, Fluorescence, Derivative Assay, Binding Assay

Journal: Regenerative Therapy

Article Title: Addition of Chk1 inhibitor and BMP4 cooperatively promotes retinal tissue formation in self-organizing human pluripotent stem cell differentiation culture

doi: 10.1016/j.reth.2021.12.003

Figure Lengend Snippet: Selectiv e induction of NR-RPE organoids with NR tissue encapsulated in RPE under treatment with 1.5 nM rhBMP4 and Chk1i PD407824 . (A) Bright-field with Rx:: Venus fluorescence image of hESC-derived 3D-retinas on day 17. Note that low-concentration (0.15 nM) rhBMP4 and PD treatment in 3D-retina efficiently promoted formation of Rx:: Venus-positive 3D-retinas compared to the 0.15 nM rhBMP4 treatment alone (control). (B) 1.5 nM rhBMP4 + PD-treated day 21 3D-retinas show self-organized, Rx:: Venus-expressing NR tissue in these pigmented spheres. (C-D) Immunostaining of day 21 NR-RPE organoids for Chx10 (NR progenitor cells; green), Mitf (RPE; red), Zo-1 (Apical; white) and Collagen-IV (basement membrane; red) with DAPI nuclear staining (blue). (C) Chx10 positive NR progenitor cells were located in the Mitf positive RPE capsule. Arrows indicate the tapering structure of the NR-RPE junction. (D) Zo-1 and Collagen-IV immunostaining show the spontaneously developed apico-basal polarity in NR-RPE organoids. (E) Immunostaining of 1.5 nM rhBMP4 + PD-treated NR-RPE organoids for Crx (photoreceptor precursor cells; green) and Chx10 (NR progenitor cells; red) on day 60. Crx positive photoreceptor precursors located and accumulated in the most apical layer facing the RPE. Scale bars, 500 μm (A, B upper image), 200 μm (B, lower image), 100 μm (C-D), 20 μm (E).

Article Snippet: SB-4 (#6881) and PD407824 (#2694) were purchased from R&D Systems.

Techniques: Fluorescence, Derivative Assay, Concentration Assay, Control, Expressing, Immunostaining, Membrane, Staining

Journal: Regenerative Therapy

Article Title: Addition of Chk1 inhibitor and BMP4 cooperatively promotes retinal tissue formation in self-organizing human pluripotent stem cell differentiation culture

doi: 10.1016/j.reth.2021.12.003

Figure Lengend Snippet: An addition of Chk1i PD407824 directs the aggregates to form spherical morphology with a distributional change in proliferating cells . (A-B) Representative hiPSC-derived aggregate images of day 6 and day17 with the indicated rhBMP4 and PD concentrations. Aggregates of all six differentiation conditions formed epithelial structure. PD-treated aggregates (+PD, 0.15 nM BMP4 + PD, 1.5 nM BMP4 + PD) show a spherical morphology compared to the non-treated condition. (C-D) Immunostaining for Ki67 (green) to analyze the distribution of proliferating cells in the day 6 and day 17 aggregates. Blue, nuclear staining with DAPI. Scale bars, 500 μm (A. upper image), 200 μm (A, lower image), 100 μm (C-D).

Article Snippet: SB-4 (#6881) and PD407824 (#2694) were purchased from R&D Systems.

Techniques: Derivative Assay, Immunostaining, Staining

Journal: Regenerative Therapy

Article Title: Addition of Chk1 inhibitor and BMP4 cooperatively promotes retinal tissue formation in self-organizing human pluripotent stem cell differentiation culture

doi: 10.1016/j.reth.2021.12.003

Figure Lengend Snippet: Chk1i PD407824 and rhBMP4 promotes phosphorylation of SMAD1/5/9 toward inside the aggregates concomitantly with NR formation . (A-B) Representative confocal images of day 6 and day17 hiPSC-derived aggregate staining for pSmad1/5/9 (green) and NR progenitor markder Chx10 (red). Blue, nuclear staining with DAPI. (C) Immunostaining for Tbx5 (green) and EphrinB2 (red), the downstream of BMP signaling during retinal development in vivo. (D-E) Apical marker Zo-1 (green) and basement membrane marker Laminin (red) immunostaining show the apico-basal polarity in day 6 and day 17 aggregates. Arrows indicate self-organized NR tissues within aggregate. Scale bars, 100 μm (C).

Article Snippet: SB-4 (#6881) and PD407824 (#2694) were purchased from R&D Systems.

Techniques: Phospho-proteomics, Derivative Assay, Staining, Immunostaining, In Vivo, Marker, Membrane

Journal: Regenerative Therapy

Article Title: Addition of Chk1 inhibitor and BMP4 cooperatively promotes retinal tissue formation in self-organizing human pluripotent stem cell differentiation culture

doi: 10.1016/j.reth.2021.12.003

Figure Lengend Snippet: Chk1i PD407824 -treated 3D-retina has the ability to produce retinal cells, retinal ganglion cells, horizontal cells, and cone and rod photoreceptor precursors . (A, B) Bright-field view with Rx:: Venus fluorescence in PD-treated or non-treated hESC-3D-retina on days 60 (A′) and 136 (B′). Note that a stratified, continuous NR epithelium was maintained in PD-treated 3D-retina during long-term maturation culture. (C-L) Immunostaining of the hESC-3D-retina with antibodies for (C, D) Rx (green) and Pmel17 (red), (E-F) Sox2 (green) and Ki67 (red) (G-H) Crx (green) and Chx10 (red) (I-J) Pax6 (green) and recoverin (red) (K, L) Rxrγ (green) and Nrl (red). Blue, nuclear staining with DAPI. (C, E, G, I, K) Day 60. (D, F, H, J, L) Day 136. Scale bars, 500 μm (A), 200 μm (B), 20 μm (E-L).

Article Snippet: SB-4 (#6881) and PD407824 (#2694) were purchased from R&D Systems.

Techniques: Fluorescence, Immunostaining, Staining