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fop hdf lines fop1 and fop2  (Coriell Institute for Medical Research)

 
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    Coriell Institute for Medical Research fop hdf lines fop1 and fop2
    Increased efficiency of hiPSC generation from <t>FOP</t> HDFs via the BMP-SMAD signaling pathway. (A and B) Number of TRA-1-60–positive colonies (A) or the ratio of TRA-1-60–positive cells (B) from reprogrammed FOP HDFs deposited in the Coriell Institute and collected at UCSF and four different normal <t>HDF</t> lines. TRA1-60–positive cells were analyzed 25 d after transfection from 10,000 cells per 100-mm dish at replating. Results are mean and SE, n = 3. *P < 0.05 (t test). (C and D) Effects of BMP-SMAD signal inhibitors, Dorsomorphin (1 µM), LDN-193189 (1 µM), P38 MAPK inhibitor, SB203580 (10 µM), and vehicle (0.1% DMSO) on hiPSC generation from <t>HDF-FOP1</t> (C) and <t>HDF-FOP2</t> (D) with OSKM retroviral transduction. ESC-like colonies were counted 25 d after the transduction from 50,000 cells per 100-mm dish at replating. Results are mean and SE, n = 3. *P < 0.05, **P < 0.01 (Dunnett’s test with vehicle conditions). (E and F) Effects of the overexpression of inhibitory SMADs SMAD6 or SMAD7 on hiPSC generation from HDF-FOP1 (E) and HDF-FOP2 (F) with OSKM retroviral transduction. ESC-like colonies were counted 25 d after transduction from 50,000 cells per 100-mm dish at replating. Results are mean and SE, n = 3. *P < 0.05, **P < 0.01 (Dunnett’s test with GFP conditions). (G and H) Effects of ACVR1 knockdown on hiPSC generation from HDF-FOP1 (G) and HDF-FOP2 (H) with episomal plasmids. TRA-1-60–positive colonies were counted 25 d after transduction from 5,000 cells per 35-mm dish at replating. Results are mean and SE, n = 3. *P < 0.05, **P < 0.01 (Dunnett’s test with nontarget siRNA conditions).
    Fop Hdf Lines Fop1 And Fop2, supplied by Coriell Institute for Medical Research, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/whale+optimization+algorithm+based+fopi+controllers/pmc05135304-737-3-11?v=Coriell+Institute+for+Medical+Research
    Average 90 stars, based on 1 article reviews
    fop hdf lines fop1 and fop2 - by Bioz Stars, 2026-08
    90/100 stars

    Images

    1) Product Images from "BMP-SMAD-ID promotes reprogramming to pluripotency by inhibiting p16/INK4A-dependent senescence"

    Article Title: BMP-SMAD-ID promotes reprogramming to pluripotency by inhibiting p16/INK4A-dependent senescence

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1603668113

    Increased efficiency of hiPSC generation from FOP HDFs via the BMP-SMAD signaling pathway. (A and B) Number of TRA-1-60–positive colonies (A) or the ratio of TRA-1-60–positive cells (B) from reprogrammed FOP HDFs deposited in the Coriell Institute and collected at UCSF and four different normal HDF lines. TRA1-60–positive cells were analyzed 25 d after transfection from 10,000 cells per 100-mm dish at replating. Results are mean and SE, n = 3. *P < 0.05 (t test). (C and D) Effects of BMP-SMAD signal inhibitors, Dorsomorphin (1 µM), LDN-193189 (1 µM), P38 MAPK inhibitor, SB203580 (10 µM), and vehicle (0.1% DMSO) on hiPSC generation from HDF-FOP1 (C) and HDF-FOP2 (D) with OSKM retroviral transduction. ESC-like colonies were counted 25 d after the transduction from 50,000 cells per 100-mm dish at replating. Results are mean and SE, n = 3. *P < 0.05, **P < 0.01 (Dunnett’s test with vehicle conditions). (E and F) Effects of the overexpression of inhibitory SMADs SMAD6 or SMAD7 on hiPSC generation from HDF-FOP1 (E) and HDF-FOP2 (F) with OSKM retroviral transduction. ESC-like colonies were counted 25 d after transduction from 50,000 cells per 100-mm dish at replating. Results are mean and SE, n = 3. *P < 0.05, **P < 0.01 (Dunnett’s test with GFP conditions). (G and H) Effects of ACVR1 knockdown on hiPSC generation from HDF-FOP1 (G) and HDF-FOP2 (H) with episomal plasmids. TRA-1-60–positive colonies were counted 25 d after transduction from 5,000 cells per 35-mm dish at replating. Results are mean and SE, n = 3. *P < 0.05, **P < 0.01 (Dunnett’s test with nontarget siRNA conditions).
    Figure Legend Snippet: Increased efficiency of hiPSC generation from FOP HDFs via the BMP-SMAD signaling pathway. (A and B) Number of TRA-1-60–positive colonies (A) or the ratio of TRA-1-60–positive cells (B) from reprogrammed FOP HDFs deposited in the Coriell Institute and collected at UCSF and four different normal HDF lines. TRA1-60–positive cells were analyzed 25 d after transfection from 10,000 cells per 100-mm dish at replating. Results are mean and SE, n = 3. *P < 0.05 (t test). (C and D) Effects of BMP-SMAD signal inhibitors, Dorsomorphin (1 µM), LDN-193189 (1 µM), P38 MAPK inhibitor, SB203580 (10 µM), and vehicle (0.1% DMSO) on hiPSC generation from HDF-FOP1 (C) and HDF-FOP2 (D) with OSKM retroviral transduction. ESC-like colonies were counted 25 d after the transduction from 50,000 cells per 100-mm dish at replating. Results are mean and SE, n = 3. *P < 0.05, **P < 0.01 (Dunnett’s test with vehicle conditions). (E and F) Effects of the overexpression of inhibitory SMADs SMAD6 or SMAD7 on hiPSC generation from HDF-FOP1 (E) and HDF-FOP2 (F) with OSKM retroviral transduction. ESC-like colonies were counted 25 d after transduction from 50,000 cells per 100-mm dish at replating. Results are mean and SE, n = 3. *P < 0.05, **P < 0.01 (Dunnett’s test with GFP conditions). (G and H) Effects of ACVR1 knockdown on hiPSC generation from HDF-FOP1 (G) and HDF-FOP2 (H) with episomal plasmids. TRA-1-60–positive colonies were counted 25 d after transduction from 5,000 cells per 35-mm dish at replating. Results are mean and SE, n = 3. *P < 0.05, **P < 0.01 (Dunnett’s test with nontarget siRNA conditions).

    Techniques Used: Transfection, Retroviral, Transduction, Over Expression, Knockdown

    FOP HDFs showed hyperactive BMP-SMAD signaling and increased efficiency of iPSC generation. (A) Direct DNA sequence analysis of 617 G (or A) in ACVR1 from genomic DNA of HDF-1323, HDF-FOP1, and HDF-FOP2. (B) Protein expression of phosphorylated SMAD1/5/8, total SMAD1/5/8, and α-tubulin in normal or FOP fibroblasts treated with 100 ng/mL BMP4 for 1 h and analyzed by Western blotting. (C) Protein expression levels of phosphorylated SMAD1/5/8 (normalized to the amount of α-tubulin expression) in normal or FOP fibroblasts. The results are mean and SE, n = 4. *P < 0.05 (Dunnett’s test with HDF-1323). (D) iPSC colonies have an ESC-like morphology with a flat, round shape and a distinct edge (Upper). Non–ESC-like colonies are epithelial with irregular edges (Lower). (Scale bars: 200 µm.) (E–H) The efficiency of ESC-like colonies from normal or FOP HDFs with retroviral transduction of OSKM in (E and G) or OSK in (F and H) at different seeding densities. Transduced cells were replated at 50,000 (E and F), 150,000, or 500,000 cells per 100-mm dish. Colony numbers were counted 25 or 30 d after transduction in OSKM or OSK conditions, respectively. Results are mean and SE, n = 4. *P < 0.05, **P < 0.01 (Dunnett’s test with HDF-1323). Hashtags (#) indicate that cells became confluent before ESC-like colonies appeared. (I) ALP staining of ESC-like colonies from FOP HDFs of three Japanese patients (FOP-J1, -J2, and -J3) and normal fibroblasts (TIG-120). The images were taken 25 d after OSKM retroviral transduction. (J and K) Numbers of ESC-like colonies from FOP HDFs of three Japanese patients with OSKM (J) and OSK (K) retroviral transduction. Colony numbers are automatically counted by ALP staining in every 5 d after transduction from 50,000 cells per 100-mm dish at replating. Results are mean and SE, n = 4.
    Figure Legend Snippet: FOP HDFs showed hyperactive BMP-SMAD signaling and increased efficiency of iPSC generation. (A) Direct DNA sequence analysis of 617 G (or A) in ACVR1 from genomic DNA of HDF-1323, HDF-FOP1, and HDF-FOP2. (B) Protein expression of phosphorylated SMAD1/5/8, total SMAD1/5/8, and α-tubulin in normal or FOP fibroblasts treated with 100 ng/mL BMP4 for 1 h and analyzed by Western blotting. (C) Protein expression levels of phosphorylated SMAD1/5/8 (normalized to the amount of α-tubulin expression) in normal or FOP fibroblasts. The results are mean and SE, n = 4. *P < 0.05 (Dunnett’s test with HDF-1323). (D) iPSC colonies have an ESC-like morphology with a flat, round shape and a distinct edge (Upper). Non–ESC-like colonies are epithelial with irregular edges (Lower). (Scale bars: 200 µm.) (E–H) The efficiency of ESC-like colonies from normal or FOP HDFs with retroviral transduction of OSKM in (E and G) or OSK in (F and H) at different seeding densities. Transduced cells were replated at 50,000 (E and F), 150,000, or 500,000 cells per 100-mm dish. Colony numbers were counted 25 or 30 d after transduction in OSKM or OSK conditions, respectively. Results are mean and SE, n = 4. *P < 0.05, **P < 0.01 (Dunnett’s test with HDF-1323). Hashtags (#) indicate that cells became confluent before ESC-like colonies appeared. (I) ALP staining of ESC-like colonies from FOP HDFs of three Japanese patients (FOP-J1, -J2, and -J3) and normal fibroblasts (TIG-120). The images were taken 25 d after OSKM retroviral transduction. (J and K) Numbers of ESC-like colonies from FOP HDFs of three Japanese patients with OSKM (J) and OSK (K) retroviral transduction. Colony numbers are automatically counted by ALP staining in every 5 d after transduction from 50,000 cells per 100-mm dish at replating. Results are mean and SE, n = 4.

    Techniques Used: Sequencing, Expressing, Western Blot, Retroviral, Transduction, Staining

    Expression of BMP-SMAD pathway genes during iPSC generation. (A) Expression levels of ID1-3, SMAD1, and ACVR1 in normal or FOP HDFs stimulated by BMP4 analyzed by RT-qPCR. Normal HDF (HDF-1323) were starved for serum overnight and then treated with 100 ng/mL BMP4 for 1 h. Results are mean and SE, n = 5. *P < 0.05, ***P < 0.001 (Dunnett’s test with HDF-1323). (B) Expression levels of ID1–3, SMAD1, EPCAM, OCLN, SNAI1, SNAI2, and ZEB1 in normal or FOP HDFs untransfected or after 5 d of transfection by epiY4 detected by RT-qPCR. Results are mean and SE, n = 3. *P < 0.05, ***P < 0.001 (t test). (C) Changes in expression levels of ID1, ID2, ID3, EPCAM, P16/INK4A, and NANOG by retroviral transduction. Normal HDFs (HDF-WTc) uninfected or after 7 d of retroviral transduction carrying O(OCT4), S(SOX2), K(KLF4), M(MYC), or their combinations were analyzed by RT-qPCR. Expression level of uninfected HDFs is defined as 1.0. Results are mean and SE, n = 3.
    Figure Legend Snippet: Expression of BMP-SMAD pathway genes during iPSC generation. (A) Expression levels of ID1-3, SMAD1, and ACVR1 in normal or FOP HDFs stimulated by BMP4 analyzed by RT-qPCR. Normal HDF (HDF-1323) were starved for serum overnight and then treated with 100 ng/mL BMP4 for 1 h. Results are mean and SE, n = 5. *P < 0.05, ***P < 0.001 (Dunnett’s test with HDF-1323). (B) Expression levels of ID1–3, SMAD1, EPCAM, OCLN, SNAI1, SNAI2, and ZEB1 in normal or FOP HDFs untransfected or after 5 d of transfection by epiY4 detected by RT-qPCR. Results are mean and SE, n = 3. *P < 0.05, ***P < 0.001 (t test). (C) Changes in expression levels of ID1, ID2, ID3, EPCAM, P16/INK4A, and NANOG by retroviral transduction. Normal HDFs (HDF-WTc) uninfected or after 7 d of retroviral transduction carrying O(OCT4), S(SOX2), K(KLF4), M(MYC), or their combinations were analyzed by RT-qPCR. Expression level of uninfected HDFs is defined as 1.0. Results are mean and SE, n = 3.

    Techniques Used: Expressing, Quantitative RT-PCR, Transfection, Retroviral, Transduction

    Expression of ID genes and p16/INK4A during iPSC generation. (A) Expression levels of ID genes in HDFs transduced by retroviral ID genes. Normal HDFs (HDF-1323 transduced with lentiviral Slc7a1) were transduced with retroviral GFP, ID1, ID2, ID3, or ID4 and cultured for 5 d. Results are mean and SE, n = 3. (B) Expression levels of ID genes in HDFs transfected with siRNAs against ID genes. Normal HDF (HDF-1323) were transfected with nontarget, ID1 no. 1, ID1 no. 2, ID2 no. 1, ID2 no. 2, ID3 no. 1, or ID3 no. 2 siRNA and cultured for 3 d. Results are mean and SE, n = 3. (C) Expression levels of Id genes in Nanog-GFP MEF transduced with retroviral Id genes. Nanog-GFP MEFs were transduced with retroviral DsRed, Id1, Id2, Id3, or Id4 and cultured for 5 d. For Id1–3 expression, results are mean and SE, n = 3. For Id4 expression, a DNA gel image of electrophoresis after 40 cycles of PCR is shown. (D) Expression levels of p16/INK4A, p15/INK4B, and p14/ARF in HDFs carrying siRNA against p16/INK4A analyzed by RT-qPCR. HDF-1323 were transfected with nontarget, p16 no. 1, p16 no. 2, p16 no. 3, or p16 no. 4 siRNA and cultured for 3 d. Results show mean and SE, n = 3. (E) Protein expression of p16/INK4A and GAPDH in HDFs carrying siRNA against P16/INK4A. OSKM-transduced HDF-1323 (at day 2) were treated with nontarget, P16 no. 1, P16 no. 2, p16 no. 3, or P16 no. 4 siRNA and cultured for 3 d and then analyzed with Western blotting. Secondary antibodies for p16/INK4A or GAPDH were labeled with IRdye680LT (red) or IRdye800CW (green), respectively. (F) Protein expression levels of p16/INK4A (normalized to the amount of GAPDH expression) in HDF-1323. Results are mean and SE, n = 4. (G and H) Cell proliferation rates of normal and FOP HDFs transduced with retroviral OSKM (G) and of normal HDFs transduced with retroviral OSKM and ID genes (H). At day 0, transduced cells (3 d after transduction) were replated at 10,000 cells per 35-mm dish and then counted daily. At day 1, the medium was changed to SNL-conditioned hESC medium. Results are mean and SE, n = 4. (I–L) p16/INK4A protein expression of FOP and normal HDFs transduced with retroviral OSKM or untransduced and HiPSCs (I and J) or of normal HDFs transduced with retroviral OSKM and ID genes or transfected with siRNA against ID genes or P16/INK4A (K and L). At day 0, transduced HDFs (3 d after transduction) were replated at 10,000 cells per 35-mm dish. At day 1, the medium was changed to SNL-conditioned hESC medium. The cells were collected for Western blot analysis at day 5. Secondary antibodies for p16/INK4A or GAPDH were labeled with IRdye680LT (red) or IRdye800CW (green), respectively. Results are mean and SE, n = 3. *P < 0.05 (t test in B), *P < 0.05, **P < 0.01 (Dunnett’s test with 4F + GFP conditions in D).
    Figure Legend Snippet: Expression of ID genes and p16/INK4A during iPSC generation. (A) Expression levels of ID genes in HDFs transduced by retroviral ID genes. Normal HDFs (HDF-1323 transduced with lentiviral Slc7a1) were transduced with retroviral GFP, ID1, ID2, ID3, or ID4 and cultured for 5 d. Results are mean and SE, n = 3. (B) Expression levels of ID genes in HDFs transfected with siRNAs against ID genes. Normal HDF (HDF-1323) were transfected with nontarget, ID1 no. 1, ID1 no. 2, ID2 no. 1, ID2 no. 2, ID3 no. 1, or ID3 no. 2 siRNA and cultured for 3 d. Results are mean and SE, n = 3. (C) Expression levels of Id genes in Nanog-GFP MEF transduced with retroviral Id genes. Nanog-GFP MEFs were transduced with retroviral DsRed, Id1, Id2, Id3, or Id4 and cultured for 5 d. For Id1–3 expression, results are mean and SE, n = 3. For Id4 expression, a DNA gel image of electrophoresis after 40 cycles of PCR is shown. (D) Expression levels of p16/INK4A, p15/INK4B, and p14/ARF in HDFs carrying siRNA against p16/INK4A analyzed by RT-qPCR. HDF-1323 were transfected with nontarget, p16 no. 1, p16 no. 2, p16 no. 3, or p16 no. 4 siRNA and cultured for 3 d. Results show mean and SE, n = 3. (E) Protein expression of p16/INK4A and GAPDH in HDFs carrying siRNA against P16/INK4A. OSKM-transduced HDF-1323 (at day 2) were treated with nontarget, P16 no. 1, P16 no. 2, p16 no. 3, or P16 no. 4 siRNA and cultured for 3 d and then analyzed with Western blotting. Secondary antibodies for p16/INK4A or GAPDH were labeled with IRdye680LT (red) or IRdye800CW (green), respectively. (F) Protein expression levels of p16/INK4A (normalized to the amount of GAPDH expression) in HDF-1323. Results are mean and SE, n = 4. (G and H) Cell proliferation rates of normal and FOP HDFs transduced with retroviral OSKM (G) and of normal HDFs transduced with retroviral OSKM and ID genes (H). At day 0, transduced cells (3 d after transduction) were replated at 10,000 cells per 35-mm dish and then counted daily. At day 1, the medium was changed to SNL-conditioned hESC medium. Results are mean and SE, n = 4. (I–L) p16/INK4A protein expression of FOP and normal HDFs transduced with retroviral OSKM or untransduced and HiPSCs (I and J) or of normal HDFs transduced with retroviral OSKM and ID genes or transfected with siRNA against ID genes or P16/INK4A (K and L). At day 0, transduced HDFs (3 d after transduction) were replated at 10,000 cells per 35-mm dish. At day 1, the medium was changed to SNL-conditioned hESC medium. The cells were collected for Western blot analysis at day 5. Secondary antibodies for p16/INK4A or GAPDH were labeled with IRdye680LT (red) or IRdye800CW (green), respectively. Results are mean and SE, n = 3. *P < 0.05 (t test in B), *P < 0.05, **P < 0.01 (Dunnett’s test with 4F + GFP conditions in D).

    Techniques Used: Expressing, Retroviral, Transduction, Cell Culture, Transfection, Electrophoresis, Quantitative RT-PCR, Western Blot, Labeling

    BMP-SMAD-ID signaling axis is functionally epistatic to p16/INK4A in iPSC generation. (A–C) Effects of p16/INK4A knockdown during reprogramming on hiPSC generation from FOP and normal HDFs (A), normal HDFs (HDF-WTc) transduced with retroviral OSKM and ID1-4 (B), or normal fibroblasts (HDF-WTc) transfected with ID2 or ID1/3 siRNA (C). TRA-1-60–positive colonies were counted 25 d after electroporation of epiY4 from 10,000 cells per 100-mm dish at replating (A and C) or after transduction with retroviral OSKM and ID1-4 from 50,000 cells per 100-mm dish at replating (B). Results are mean and SE, n = 3. *P < 0.05, **P < 0.01, ***P < 0.001 (Dunnett’s test with each siRNA NT condition). (D) Effects of overexpressed Id genes on generating miPSCs from p16 (+/+), p16 (+/−) MEF, or p16 (−/−) MEFs (crossed with Nanog-GFP) with OSK retroviral transduction. Nanog-GFP–positive colonies were counted at 16 d after transduction from 10,000 cells per 35-mm dish at replating. Results are mean and SE, n = 4. **P < 0.01 (Dunnett’s test with each +DsRed condition).
    Figure Legend Snippet: BMP-SMAD-ID signaling axis is functionally epistatic to p16/INK4A in iPSC generation. (A–C) Effects of p16/INK4A knockdown during reprogramming on hiPSC generation from FOP and normal HDFs (A), normal HDFs (HDF-WTc) transduced with retroviral OSKM and ID1-4 (B), or normal fibroblasts (HDF-WTc) transfected with ID2 or ID1/3 siRNA (C). TRA-1-60–positive colonies were counted 25 d after electroporation of epiY4 from 10,000 cells per 100-mm dish at replating (A and C) or after transduction with retroviral OSKM and ID1-4 from 50,000 cells per 100-mm dish at replating (B). Results are mean and SE, n = 3. *P < 0.05, **P < 0.01, ***P < 0.001 (Dunnett’s test with each siRNA NT condition). (D) Effects of overexpressed Id genes on generating miPSCs from p16 (+/+), p16 (+/−) MEF, or p16 (−/−) MEFs (crossed with Nanog-GFP) with OSK retroviral transduction. Nanog-GFP–positive colonies were counted at 16 d after transduction from 10,000 cells per 35-mm dish at replating. Results are mean and SE, n = 4. **P < 0.01 (Dunnett’s test with each +DsRed condition).

    Techniques Used: Knockdown, Transduction, Retroviral, Transfection, Electroporation



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