ksr xf  (Thermo Fisher)


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    Name:
    CTS KnockOut SR XenoFree Medium
    Description:
    GIBCO CTS KnockOut SR XenoFree Medium when formulated as a complete medium enables the growth and expansion of human embryonic stem cells hESC and human induced pluripotent stem cells hiPSC in a cell culture medium containing only human derived or human recombinant proteins to facilitate the transition of hESC and hiPSC research from the bench to the clinic The CTS product line enables you to reduce your burden in qualifying reagents during your transition from research applications to clinical applications • Defined xenofree medium for hESC⁄iPSC stem cell culture • First to market xenofree hESC⁄iPSC product intended for Ex Vivo tissue and cell culture processing applications CTS KnockOut SR XenoFree Medium does not contain bovine or other non human animal derived components Besides pluripotent stem cell culture expansion and maintenance CTS KnockOut SR XenoFree Medium can be used for hESC⁄hiPSC cryopreservation derivation and differentiation studies GIBCO CTS products are of high quality and are supplied with harmonized documentation such as Certificates of Analysis Certificates of Origin and access to authorization letters for our Drug Master Files as appropriate For human ex vivo tissue and cell culture processing applications CAUTION Not intended for direct administration to humans or animals
    Catalog Number:
    12618013
    Price:
    None
    Category:
    Cell Culture Transfection Reagents
    Applications:
    Cell Culture|Cell Therapy Systems|Clinical|Clinical & Translational Research|Embryonic Stem Cell Culture|Induced Pluripotent Stem Cell Culture|Mammalian Cell Culture|Stem Cell Culture|Stem Cell Research|Stem Cell Therapy Research
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    Structured Review

    Thermo Fisher ksr xf
    GIBCO CTS KnockOut SR XenoFree Medium when formulated as a complete medium enables the growth and expansion of human embryonic stem cells hESC and human induced pluripotent stem cells hiPSC in a cell culture medium containing only human derived or human recombinant proteins to facilitate the transition of hESC and hiPSC research from the bench to the clinic The CTS product line enables you to reduce your burden in qualifying reagents during your transition from research applications to clinical applications • Defined xenofree medium for hESC⁄iPSC stem cell culture • First to market xenofree hESC⁄iPSC product intended for Ex Vivo tissue and cell culture processing applications CTS KnockOut SR XenoFree Medium does not contain bovine or other non human animal derived components Besides pluripotent stem cell culture expansion and maintenance CTS KnockOut SR XenoFree Medium can be used for hESC⁄hiPSC cryopreservation derivation and differentiation studies GIBCO CTS products are of high quality and are supplied with harmonized documentation such as Certificates of Analysis Certificates of Origin and access to authorization letters for our Drug Master Files as appropriate For human ex vivo tissue and cell culture processing applications CAUTION Not intended for direct administration to humans or animals
    https://www.bioz.com/result/ksr xf/product/Thermo Fisher
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    ksr xf - by Bioz Stars, 2021-03
    94/100 stars

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    Related Articles

    Knock-Out:

    Article Title: Xeno- and feeder-free differentiation of human pluripotent stem cells to two distinct ocular epithelial cell types using simple modifications of one method
    Article Snippet: .. Undifferentiated hPSCs were detached and transferred to Corning® Costar® Ultra-Low attachment plates in XF-Ko-SR medium (KnockOut™ Dulbecoo’s modified Eagle’s medium (DMEM) supplemented with 15% KnockOut™ SR XenoFree CTS™ (XF-Ko-SR), 2 mM GlutaMAX™, 0.1 mM 2-mercaptoethanol, 1% MEM non-essential amino acids, and 50 U/ml penicillin-streptomycin (all from Gibco, Thermo Fisher Scientific)) supplemented with 5 μM or 10 μM blebbistatin (Sigma-Aldrich) or Rock inhibitor Y-27632 dihydrochloride (ROCKi; R & D Systems) to induce embryoid body (EB) formation overnight at +37 °C. .. For RPE differentiation, EBs were either allowed to undergo spontaneous differentiation in 15% XF-Ko-SR medium, or were subjected to neuroectodermal induction with 10 μM SB-505124 (Sigma-Aldrich) and 10 μM IWP-2 (Merck Millipore).

    Article Title: Evaluation of Therapeutic Oligonucleotides for Familial Amyloid Polyneuropathy in Patient-Derived Hepatocyte-Like Cells
    Article Snippet: After 24 hours, culture medium was changed for the next 3 days to DMEM/F12 supplemented with 100 ng/ml recombinant Activin-A (R & D Systems), 100 ng/ml fibroblast growth factor-2 (FGF2, Peprotech), 50 ng/ml recombinant human Wnt3a (R & D Systems). .. Concentration of KnockOut SR Xenofree CTS (KSR, Gibco) was 0%, 0.2% and 2.0% for the first, second and third day, respectively. .. For the next 8 days, cells were cultivated in DMEM/F12 supplemented with 10% KSR, 1mM NEAA, 1mM L-Glutamine, 1% dimethyl sulfoxide (DMSO, Sigma-Aldrich), and 100 ng/ml hepatocyte growth factor (HGF, Peprotech).

    Article Title: Assembly and Function of a Bioengineered Human Liver for Transplantation Generated Solely from Induced Pluripotent Stem Cells
    Article Snippet: At the end of Stage 3, cells were detached and either re-plated at a 30%–40% confluence in 3D sandwich culture or seeded into decellularized liver matrix for further maturation. .. Cells were grown for 4 days in a defined medium containing 45% DMEM low glucose 1g/l (ThermoFisher Scientific, Waltham, MA), 45% F-12 (ThermoFisher Scientific, Waltham, MA), 10% CTS KnockOut SR XenoFree Medium, 0.5% Non-Essential Amino Acids (ThermoFisher Scientific, Waltham, MA), 0.5% L-glutamine (ThermoFisher Scientific, Waltham, MA), 0.1% of Gentamicin/Amphotericin-B (ThermoFisher Scientific, Waltham, MA), 1% of Pennicillin/Streptomycin (ThermoFisher Scientific, Waltham, MA), 50 ng/ml HGF (Kindly provided by George Michalopoulos), 1% DMSO, 0.5uM Dexamethasone (Sigma-Aldrich, Saint Louis, MO), 0.1% of Ascorbic Acid (Sigma-Aldrich, Saint Louis, MO), 0.1% of Bovine Serum Albumin Free of Fatty Acids, 0.1% of Hydrocortisone, 0.1% of Transferrin, 0.1% of Insulin (HCM Bullet Kit, ThermoFisher Scientific, Waltham, MA), 100uM of Urso deoxycolic acid (Sigma-Aldrich, Saint Louis, MO), 20uM of Palmitic Acid (Sigma-Aldrich, Saint Louis, MO), 30 uM of Oleic Acid (Sigma-Aldrich, Saint Louis, MO), 20 uM of Rifampicin (Sigma-Aldrich, Saint Louis, Missouri) and 1x of Cholesterol (ThermoFisher Scientific, Waltham, MA) (Stage 4, hepatic maturation). ..

    Article Title: A short-term plastic adherence incubation of the stromal vascular fraction leads to a predictable GMP-compliant cell-product
    Article Snippet: The resulting cells were washed twice with PBS w/o Mg2+ Ca2+ (Biochrom, Berlin, Germany), 2% KnockOut™ (ThermoFisher Scientific, Waltham, USA). .. For short-term incubation and purification, the cells were seeded on cell culture dishes (Greiner Bio-One) at high density (≥ 1.0 x 105 cells/cm2 ) in DMEM-F12, 2% KnockOut™ SR XenoFree Medium (ThermoFisher Scientific, Waltham, USA) and incubated for 16-20 hours (37°C, 6% CO2 , 95% RH). .. After that time, the cells were trypsinized (Trypsin-EDTA solution, Sigma-Aldrich, Munich, Germany) and suspended in PBS w/o Mg2+ Ca2+ (Biochrom, Berlin, Germany), 2% KnockOut™ (ThermoFisher Scientific, Waltham, USA).

    Article Title: A short-term plastic adherence incubation of the stromal vascular fraction leads to a predictable GMP-compliant cell-product
    Article Snippet: The digestion was stopped with EDTA (Corning Inc., New York , USA) as recommended by Nordmark for Collagenase digestion. .. The cell pellet was resuspended in DMEM-F12, 2% KnockOut™ SR XenoFree Medium (ThermoFisher Scientific, Waltham, USA) and filtered through different strainers (Steriflip® , Merck Millipore, Darmstadt, Germany). .. Afterward, the cells were further separated by density gradient centrifugation with Ficoll-Paque PremiumTM (400 g, 24°C, 30 minutes, GE Healthcare Bio-Sciences, Buckinghamshire, UK) instead of red blood cell (RBC) lysis.

    Modification:

    Article Title: Xeno- and feeder-free differentiation of human pluripotent stem cells to two distinct ocular epithelial cell types using simple modifications of one method
    Article Snippet: .. Undifferentiated hPSCs were detached and transferred to Corning® Costar® Ultra-Low attachment plates in XF-Ko-SR medium (KnockOut™ Dulbecoo’s modified Eagle’s medium (DMEM) supplemented with 15% KnockOut™ SR XenoFree CTS™ (XF-Ko-SR), 2 mM GlutaMAX™, 0.1 mM 2-mercaptoethanol, 1% MEM non-essential amino acids, and 50 U/ml penicillin-streptomycin (all from Gibco, Thermo Fisher Scientific)) supplemented with 5 μM or 10 μM blebbistatin (Sigma-Aldrich) or Rock inhibitor Y-27632 dihydrochloride (ROCKi; R & D Systems) to induce embryoid body (EB) formation overnight at +37 °C. .. For RPE differentiation, EBs were either allowed to undergo spontaneous differentiation in 15% XF-Ko-SR medium, or were subjected to neuroectodermal induction with 10 μM SB-505124 (Sigma-Aldrich) and 10 μM IWP-2 (Merck Millipore).

    Staining:

    Article Title: Optimized Approaches for the Induction of Putative Canine Induced Pluripotent Stem Cells from Old Fibroblasts Using Synthetic RNAs
    Article Snippet: Putative ciPSC colonies were cultured in stage 3 medium containing 10 ng/mL of bFGF (Bio-Bud) or 1000 units/mL of murine leukemia inhibitory factor (mLIF; ESGRO, Millipore, Billerica, MA, USA), plus 0.5 μM of MEK1/2 inhibitor (PD0325901; Stemgent, Cambridge, MA, USA) and 3 μM of GSK3β inhibitor (CHIR99021; Stemgent). .. Primary iPSC Colony Staining with Alkaline Phosphatase (AP) and TRA-1-60ciPSCs were washed with PBS three times after removing the iPSC culture medium from each 6-well plate, after which they were fixed in PBS containing 4% paraformaldehyde for 5 min at room temperature. .. Fixed cells were washed three times in PBS and stained with 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium (BCIP/NBT; Roche, Basel, Switzerland) solution for 30 min at room temperature in the dark.

    Concentration Assay:

    Article Title: Evaluation of Therapeutic Oligonucleotides for Familial Amyloid Polyneuropathy in Patient-Derived Hepatocyte-Like Cells
    Article Snippet: After 24 hours, culture medium was changed for the next 3 days to DMEM/F12 supplemented with 100 ng/ml recombinant Activin-A (R & D Systems), 100 ng/ml fibroblast growth factor-2 (FGF2, Peprotech), 50 ng/ml recombinant human Wnt3a (R & D Systems). .. Concentration of KnockOut SR Xenofree CTS (KSR, Gibco) was 0%, 0.2% and 2.0% for the first, second and third day, respectively. .. For the next 8 days, cells were cultivated in DMEM/F12 supplemented with 10% KSR, 1mM NEAA, 1mM L-Glutamine, 1% dimethyl sulfoxide (DMSO, Sigma-Aldrich), and 100 ng/ml hepatocyte growth factor (HGF, Peprotech).

    Incubation:

    Article Title: A short-term plastic adherence incubation of the stromal vascular fraction leads to a predictable GMP-compliant cell-product
    Article Snippet: The resulting cells were washed twice with PBS w/o Mg2+ Ca2+ (Biochrom, Berlin, Germany), 2% KnockOut™ (ThermoFisher Scientific, Waltham, USA). .. For short-term incubation and purification, the cells were seeded on cell culture dishes (Greiner Bio-One) at high density (≥ 1.0 x 105 cells/cm2 ) in DMEM-F12, 2% KnockOut™ SR XenoFree Medium (ThermoFisher Scientific, Waltham, USA) and incubated for 16-20 hours (37°C, 6% CO2 , 95% RH). .. After that time, the cells were trypsinized (Trypsin-EDTA solution, Sigma-Aldrich, Munich, Germany) and suspended in PBS w/o Mg2+ Ca2+ (Biochrom, Berlin, Germany), 2% KnockOut™ (ThermoFisher Scientific, Waltham, USA).

    Purification:

    Article Title: A short-term plastic adherence incubation of the stromal vascular fraction leads to a predictable GMP-compliant cell-product
    Article Snippet: The resulting cells were washed twice with PBS w/o Mg2+ Ca2+ (Biochrom, Berlin, Germany), 2% KnockOut™ (ThermoFisher Scientific, Waltham, USA). .. For short-term incubation and purification, the cells were seeded on cell culture dishes (Greiner Bio-One) at high density (≥ 1.0 x 105 cells/cm2 ) in DMEM-F12, 2% KnockOut™ SR XenoFree Medium (ThermoFisher Scientific, Waltham, USA) and incubated for 16-20 hours (37°C, 6% CO2 , 95% RH). .. After that time, the cells were trypsinized (Trypsin-EDTA solution, Sigma-Aldrich, Munich, Germany) and suspended in PBS w/o Mg2+ Ca2+ (Biochrom, Berlin, Germany), 2% KnockOut™ (ThermoFisher Scientific, Waltham, USA).

    Cell Culture:

    Article Title: A short-term plastic adherence incubation of the stromal vascular fraction leads to a predictable GMP-compliant cell-product
    Article Snippet: The resulting cells were washed twice with PBS w/o Mg2+ Ca2+ (Biochrom, Berlin, Germany), 2% KnockOut™ (ThermoFisher Scientific, Waltham, USA). .. For short-term incubation and purification, the cells were seeded on cell culture dishes (Greiner Bio-One) at high density (≥ 1.0 x 105 cells/cm2 ) in DMEM-F12, 2% KnockOut™ SR XenoFree Medium (ThermoFisher Scientific, Waltham, USA) and incubated for 16-20 hours (37°C, 6% CO2 , 95% RH). .. After that time, the cells were trypsinized (Trypsin-EDTA solution, Sigma-Aldrich, Munich, Germany) and suspended in PBS w/o Mg2+ Ca2+ (Biochrom, Berlin, Germany), 2% KnockOut™ (ThermoFisher Scientific, Waltham, USA).

    Recombinant:

    Article Title: CD133-enriched Xeno-Free human embryonic-derived neural stem cells expand rapidly in culture and do not form teratomas in immunodeficient mice
    Article Snippet: Human embryonic and neural stem cell culture and differentiation Culture of hESC lines Shef3, Shef4, and Shef6 (University of Sheffield, UK) was established at UC Irvine in accordance with all appropriate hSCRO and IBC protocols on mitotically-inactivated mouse embryonic fibroblasts (MEFs, EMD Millipore) and in defined media consisting of KO DMEM/ F12, 20% KO Serum Replacement (KO SR), 0.1 mM NEAA, 2 mM GlutaMAX, 0.1 mM β-Mercaptoethanol, and 20 ng/mL bFGF (All from Life Technologies). .. To transition cells to Xeno-Free (XF) culture conditions, all non-human animal-based components (MEFs, KOSR) were removed and replaced with human-based or recombinant alternatives including CELLstart CTS, KO SR Xeno-Free CTS, and KO SR GF Cocktail CTS (All from Life Technologies). ..

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  • 99
    Thermo Fisher dulbecco s modified eagle s medium dmem ksr xf ksr xf knockout dmem medium
    Stage 1: induction of eye field from human embryonic stem cells (hESC). (A): Two days’ postseeding, SHEF1 hESC were treated with <t>Dulbecco's</t> modified <t>Eagle's</t> medium <t>KSR‐XF</t> alone (Control) or Induction Medium 1 for 4 days, followed by Induction Medium 2 for 3 days (BMP4/7). Representative images showing immunocytochemistry for the pluripotency marker OCT4 and eye field markers LHX2, SOX11, PAX6, and MITF are shown. Dotted squares indicate the region that is magnified in the adjacent panels. Images are captured at ×10 magnification. (B): Quantitative polymerase chain reaction was carried out to measure expression of Mitf transcript in SHEF1 hESC treated with Induction Medium 1 alone (Control) or Induction Medium 1 supplemented with BMP4/7 (50 ng/ml, 100 ng/ml, 200 ng/ml; shown by triangle in order of ascending concentration); BMP4 (200 ng/ml); or BMP7 (200 ng/ml) ( n = 3, ±SD). Abbreviation: BMP, bone morphogenetic protein.
    Dulbecco S Modified Eagle S Medium Dmem Ksr Xf Ksr Xf Knockout Dmem Medium, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dulbecco s modified eagle s medium dmem ksr xf ksr xf knockout dmem medium/product/Thermo Fisher
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    dulbecco s modified eagle s medium dmem ksr xf ksr xf knockout dmem medium - by Bioz Stars, 2021-03
    99/100 stars
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    99
    Thermo Fisher xf ksr
    Stage 1: induction of eye field from human embryonic stem cells (hESC). (A): Two days’ postseeding, SHEF1 hESC were treated with <t>Dulbecco's</t> modified <t>Eagle's</t> medium <t>KSR‐XF</t> alone (Control) or Induction Medium 1 for 4 days, followed by Induction Medium 2 for 3 days (BMP4/7). Representative images showing immunocytochemistry for the pluripotency marker OCT4 and eye field markers LHX2, SOX11, PAX6, and MITF are shown. Dotted squares indicate the region that is magnified in the adjacent panels. Images are captured at ×10 magnification. (B): Quantitative polymerase chain reaction was carried out to measure expression of Mitf transcript in SHEF1 hESC treated with Induction Medium 1 alone (Control) or Induction Medium 1 supplemented with BMP4/7 (50 ng/ml, 100 ng/ml, 200 ng/ml; shown by triangle in order of ascending concentration); BMP4 (200 ng/ml); or BMP7 (200 ng/ml) ( n = 3, ±SD). Abbreviation: BMP, bone morphogenetic protein.
    Xf Ksr, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/xf ksr/product/Thermo Fisher
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    xf ksr - by Bioz Stars, 2021-03
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    95
    Thermo Fisher xf ksr human embryonic stem cell hesc medium
    Stage 1: induction of eye field from human embryonic stem cells (hESC). (A): Two days’ postseeding, SHEF1 hESC were treated with <t>Dulbecco's</t> modified <t>Eagle's</t> medium <t>KSR‐XF</t> alone (Control) or Induction Medium 1 for 4 days, followed by Induction Medium 2 for 3 days (BMP4/7). Representative images showing immunocytochemistry for the pluripotency marker OCT4 and eye field markers LHX2, SOX11, PAX6, and MITF are shown. Dotted squares indicate the region that is magnified in the adjacent panels. Images are captured at ×10 magnification. (B): Quantitative polymerase chain reaction was carried out to measure expression of Mitf transcript in SHEF1 hESC treated with Induction Medium 1 alone (Control) or Induction Medium 1 supplemented with BMP4/7 (50 ng/ml, 100 ng/ml, 200 ng/ml; shown by triangle in order of ascending concentration); BMP4 (200 ng/ml); or BMP7 (200 ng/ml) ( n = 3, ±SD). Abbreviation: BMP, bone morphogenetic protein.
    Xf Ksr Human Embryonic Stem Cell Hesc Medium, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/xf ksr human embryonic stem cell hesc medium/product/Thermo Fisher
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    xf ksr human embryonic stem cell hesc medium - by Bioz Stars, 2021-03
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    Image Search Results


    Stage 1: induction of eye field from human embryonic stem cells (hESC). (A): Two days’ postseeding, SHEF1 hESC were treated with Dulbecco's modified Eagle's medium KSR‐XF alone (Control) or Induction Medium 1 for 4 days, followed by Induction Medium 2 for 3 days (BMP4/7). Representative images showing immunocytochemistry for the pluripotency marker OCT4 and eye field markers LHX2, SOX11, PAX6, and MITF are shown. Dotted squares indicate the region that is magnified in the adjacent panels. Images are captured at ×10 magnification. (B): Quantitative polymerase chain reaction was carried out to measure expression of Mitf transcript in SHEF1 hESC treated with Induction Medium 1 alone (Control) or Induction Medium 1 supplemented with BMP4/7 (50 ng/ml, 100 ng/ml, 200 ng/ml; shown by triangle in order of ascending concentration); BMP4 (200 ng/ml); or BMP7 (200 ng/ml) ( n = 3, ±SD). Abbreviation: BMP, bone morphogenetic protein.

    Journal: Stem Cells Translational Medicine

    Article Title: Directing Differentiation of Pluripotent Stem Cells Toward Retinal Pigment Epithelium Lineage

    doi: 10.5966/sctm.2016-0088

    Figure Lengend Snippet: Stage 1: induction of eye field from human embryonic stem cells (hESC). (A): Two days’ postseeding, SHEF1 hESC were treated with Dulbecco's modified Eagle's medium KSR‐XF alone (Control) or Induction Medium 1 for 4 days, followed by Induction Medium 2 for 3 days (BMP4/7). Representative images showing immunocytochemistry for the pluripotency marker OCT4 and eye field markers LHX2, SOX11, PAX6, and MITF are shown. Dotted squares indicate the region that is magnified in the adjacent panels. Images are captured at ×10 magnification. (B): Quantitative polymerase chain reaction was carried out to measure expression of Mitf transcript in SHEF1 hESC treated with Induction Medium 1 alone (Control) or Induction Medium 1 supplemented with BMP4/7 (50 ng/ml, 100 ng/ml, 200 ng/ml; shown by triangle in order of ascending concentration); BMP4 (200 ng/ml); or BMP7 (200 ng/ml) ( n = 3, ±SD). Abbreviation: BMP, bone morphogenetic protein.

    Article Snippet: On day 2, cells were switched to Induction Medium 1: Dulbecco's modified Eagle's medium (DMEM) KSR‐XF (KnockOut DMEM medium [Thermo Fisher], supplemented with 20% KnockOut Serum Replacement Xeno‐Free [Thermo Fisher], 1% β‐Mercaptoethanol [Sigma‐Aldrich], 1% GlutaMax [Thermo Fisher], and 1% non‐essential amino acid solution [Thermo Fisher]), supplemented with two inhibitors (LDN/SB): 1 μM LDN‐193189 (Stemgent, Cambridge, MA, https://www.stemgent.com ) and 10 µM SB‐431542 (Sigma‐Aldrich).

    Techniques: Modification, Immunocytochemistry, Marker, Real-time Polymerase Chain Reaction, Expressing, Concentration Assay

    Stage 2: Generation of a mixed RPE population. (A): Two days’ postseeding, SHEF1 human embryonic stem cells were treated with Induction Medium 1 for 4 days, followed by Induction Medium 2 for 3 days. At day 9, cells were replated in Dulbecco's modified Eagle's medium (DMEM) KSR‐XF alone (Control) or Induction Medium 3 (ActA) for 19 days. Quantitative polymerase chain reaction was used to measure expression of a panel of RPE markers. RPE generated by using spontaneous differentiation (Sp. RPE) were used for comparison ( n = 3, ±SD). (B): Representative images showing immunocytochemistry for CRALBP and MERTK in cells derived by directed differentiation and cultured as in panel A. Dotted squares indicate the region that is magnified in the panels below. RPE generated by spontaneous differentiation were used for comparison. Images are captured at ×10 magnification. (C): Quantification of CRALBP immunocytochemistry at day 28 in which cells during stage 2 were treated with DMEM KSR‐XF (Control) or Induction Medium 3 (ActA) for 3 days, 5 days, 10 days, or 19 days. For fewer than 19‐day ActA treatments, DMEM KSR‐XF was used for the remainder of the 19‐day period. RPE derived by using spontaneous differentiation (Sp. RPE) were used for comparison ( n = 3, ±SD). Abbreviations: ActA, Activin A; d, days; RPE, retinal pigment epithelium; Sp. RPE, spontaneous differentiation retinal pigment epithelium.

    Journal: Stem Cells Translational Medicine

    Article Title: Directing Differentiation of Pluripotent Stem Cells Toward Retinal Pigment Epithelium Lineage

    doi: 10.5966/sctm.2016-0088

    Figure Lengend Snippet: Stage 2: Generation of a mixed RPE population. (A): Two days’ postseeding, SHEF1 human embryonic stem cells were treated with Induction Medium 1 for 4 days, followed by Induction Medium 2 for 3 days. At day 9, cells were replated in Dulbecco's modified Eagle's medium (DMEM) KSR‐XF alone (Control) or Induction Medium 3 (ActA) for 19 days. Quantitative polymerase chain reaction was used to measure expression of a panel of RPE markers. RPE generated by using spontaneous differentiation (Sp. RPE) were used for comparison ( n = 3, ±SD). (B): Representative images showing immunocytochemistry for CRALBP and MERTK in cells derived by directed differentiation and cultured as in panel A. Dotted squares indicate the region that is magnified in the panels below. RPE generated by spontaneous differentiation were used for comparison. Images are captured at ×10 magnification. (C): Quantification of CRALBP immunocytochemistry at day 28 in which cells during stage 2 were treated with DMEM KSR‐XF (Control) or Induction Medium 3 (ActA) for 3 days, 5 days, 10 days, or 19 days. For fewer than 19‐day ActA treatments, DMEM KSR‐XF was used for the remainder of the 19‐day period. RPE derived by using spontaneous differentiation (Sp. RPE) were used for comparison ( n = 3, ±SD). Abbreviations: ActA, Activin A; d, days; RPE, retinal pigment epithelium; Sp. RPE, spontaneous differentiation retinal pigment epithelium.

    Article Snippet: On day 2, cells were switched to Induction Medium 1: Dulbecco's modified Eagle's medium (DMEM) KSR‐XF (KnockOut DMEM medium [Thermo Fisher], supplemented with 20% KnockOut Serum Replacement Xeno‐Free [Thermo Fisher], 1% β‐Mercaptoethanol [Sigma‐Aldrich], 1% GlutaMax [Thermo Fisher], and 1% non‐essential amino acid solution [Thermo Fisher]), supplemented with two inhibitors (LDN/SB): 1 μM LDN‐193189 (Stemgent, Cambridge, MA, https://www.stemgent.com ) and 10 µM SB‐431542 (Sigma‐Aldrich).

    Techniques: Modification, Real-time Polymerase Chain Reaction, Expressing, Generated, Immunocytochemistry, Derivative Assay, Cell Culture

    Stage 3: Generation of a homogenous and functional retinal pigment epithelium (RPE) population. (A): Two days’ postseeding, SHEF1 human embryonic stem cells were treated with Induction Medium 1 for 4 days, followed by Induction Medium 2 for 3 days. At day 9, cells were replated in Induction Medium 3 for a period of 19 days. At day 28, cells were replated in Dulbecco's modified Eagle's medium KSR‐XF and cultured for a period of 14 days. Representative images showing immunocytochemistry for indicated RPE markers are shown. Images are captured at ×10 magnification. (B): Cells cultured as in panel A were further dissociated and replated on transwells and cultured for a period of 10 weeks. A representative image showing en face immunocytochemistry for ZO‐1 is shown. Images are captured at ×20 magnification. (C): Spent medium from transwells described in panel B was collected from the top and bottom chambers and quantified for vascular endothelial growth factor (VEGF) and pigment epithelium‐derived factor (PEDF) concentration. The ratio of [VEGF]:[PEDF] was quantified in media from the two compartments ( n = 3, ±SD). (D): Representative confocal images showing phagocytosis of fluorescent bead (red) by RPE. ZO‐1 immunocytochemistry (green) shows the cell edge, and the presence of bead within the cell boundary indicates internalization by phagocytosis. Images are captured at ×63 magnification. Abbreviations: PEDF, pigment epithelium‐derived factor; VEGF, vascular endothelial growth factor.

    Journal: Stem Cells Translational Medicine

    Article Title: Directing Differentiation of Pluripotent Stem Cells Toward Retinal Pigment Epithelium Lineage

    doi: 10.5966/sctm.2016-0088

    Figure Lengend Snippet: Stage 3: Generation of a homogenous and functional retinal pigment epithelium (RPE) population. (A): Two days’ postseeding, SHEF1 human embryonic stem cells were treated with Induction Medium 1 for 4 days, followed by Induction Medium 2 for 3 days. At day 9, cells were replated in Induction Medium 3 for a period of 19 days. At day 28, cells were replated in Dulbecco's modified Eagle's medium KSR‐XF and cultured for a period of 14 days. Representative images showing immunocytochemistry for indicated RPE markers are shown. Images are captured at ×10 magnification. (B): Cells cultured as in panel A were further dissociated and replated on transwells and cultured for a period of 10 weeks. A representative image showing en face immunocytochemistry for ZO‐1 is shown. Images are captured at ×20 magnification. (C): Spent medium from transwells described in panel B was collected from the top and bottom chambers and quantified for vascular endothelial growth factor (VEGF) and pigment epithelium‐derived factor (PEDF) concentration. The ratio of [VEGF]:[PEDF] was quantified in media from the two compartments ( n = 3, ±SD). (D): Representative confocal images showing phagocytosis of fluorescent bead (red) by RPE. ZO‐1 immunocytochemistry (green) shows the cell edge, and the presence of bead within the cell boundary indicates internalization by phagocytosis. Images are captured at ×63 magnification. Abbreviations: PEDF, pigment epithelium‐derived factor; VEGF, vascular endothelial growth factor.

    Article Snippet: On day 2, cells were switched to Induction Medium 1: Dulbecco's modified Eagle's medium (DMEM) KSR‐XF (KnockOut DMEM medium [Thermo Fisher], supplemented with 20% KnockOut Serum Replacement Xeno‐Free [Thermo Fisher], 1% β‐Mercaptoethanol [Sigma‐Aldrich], 1% GlutaMax [Thermo Fisher], and 1% non‐essential amino acid solution [Thermo Fisher]), supplemented with two inhibitors (LDN/SB): 1 μM LDN‐193189 (Stemgent, Cambridge, MA, https://www.stemgent.com ) and 10 µM SB‐431542 (Sigma‐Aldrich).

    Techniques: Functional Assay, Modification, Cell Culture, Immunocytochemistry, Derivative Assay, Concentration Assay