dulbecco s modified eagle s medium dmem ksr xf ksr xf knockout dmem medium  (Thermo Fisher)


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    Name:
    KnockOut DMEM
    Description:
    KnockOut D MEM is a basal medium optimized for growth of undifferentiated embryonic and induced pluripotent stem cells 1 The osmolarity is optimized to approximate that of mouse embryonic tissue Contains no L glutamine
    Catalog Number:
    10829018
    Price:
    None
    Category:
    Cell Culture Transfection Reagents
    Applications:
    Cell Culture|Embryonic Stem Cell Culture|Induced Pluripotent Stem Cell Culture|Mammalian Cell Culture|Mesenchymal Stem Cell Culture|Stem Cell Culture|Stem Cell Research
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    Structured Review

    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.
    KnockOut D MEM is a basal medium optimized for growth of undifferentiated embryonic and induced pluripotent stem cells 1 The osmolarity is optimized to approximate that of mouse embryonic tissue Contains no L glutamine
    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, 2021-04
    99/100 stars

    Images

    1) Product Images from "Directing Differentiation of Pluripotent Stem Cells Toward Retinal Pigment Epithelium Lineage"

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

    Journal: Stem Cells Translational Medicine

    doi: 10.5966/sctm.2016-0088

    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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

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

    Related Articles

    Knock-Out:

    Article Title: Aldehyde dehydrogenase 1 positive glioblastoma cells show brain tumor stem cell capacity
    Article Snippet: The human GBM cell lines LN18, LN229, LNZ308, and G139 were cultured in the Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS), 2 mM l -glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin (Invitrogen) under the standard cell culture conditions at 37°C and 5% CO2 . .. To investigate the neurosphere formation, the cells were grown in a serum-free neurobasal medium consisting of Knockout DMEM (Invitrogen), Nutrient Mixture Ham's F12 + l -glutamine (Invitrogen), and 10% Knockout-Serum Replacement (Invitrogen) with 1% N2-Supplement (Invitrogen), 1% Non-Essential Amino-Acids (Invitrogen), 0.1% natural mouse laminin (Invitrogen), 2 mM l -glutamine (Biochrom), 100 U/mL penicillin (Biochrom), 100 µg/mL streptomycin (Biochrom), 10 ng/mL basic fibroblast growth factor (bFGF; Invitrogen), and 50 ng/mL epidermal growth factor (EGF; human recombinant, Millipore). .. For nonadherent growth conditions, the culture dishes were covered with 0.1% gelatin type A (Sigma-Aldrich).

    Article Title: Vascular Smooth Muscle Cells Derived from Inbred Swine Induced Pluripotent Stem Cells for Vascular Tissue Engineering
    Article Snippet: On day 0, fibroblast medium was replaced with medium containing lentiviral particles (1 mL fresh fibroblast medium mixed with 1.4 mL viral supernatant containing hSTEMCCA and 0.6 mL supernatant containing rtTA, plus 5 μg/mL polybrene (SigmaAldrich)). .. 48 hours after the first infection, medium was switched to pluripotency-promoting medium (Knockout DMEM (ThermoFisher), 10% Knockout Serum Replacement (KOSR, ThermoFisher), 10% (v/v) FBS, 20 ng/mL human leukemia growth factor (LIF, Peprotech), 20 ng/mL human basic fibroblast growth factor (bFGF, SigmaAldrich), 2 μg/mL doxycycline (Stemgent), 1% (v/v) NEAA, 1% (v/v) pen/strep, 2 mM L-glutamine and 0.1 mM β-mercaptoethanol (SigmaAldrich)) for 48 hours. .. Then the infected SEFs were dissociated on day 4 with 0.05% trypsin EDTA (SigmaAldrich) and seeded onto irradiated mouse embryonic fibroblasts (MEFs, 40,000 cells/cm2 as feeder layer) at 4,000 cells/cm2 , and cultured in siPSC medium (Dulbecco’s Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F12, ThermoFisher), 20% (v/v) KOSR, 20 ng/mL LIF, 20 ng/mL bFGF, 2 μg/mL doxycycline, 2 mM L-glutamine, 1% (v/v) NEAA, 1% (v/v) pen/strep and 0.1 mM β-mercaptoethanol).

    Article Title: Idiopathic autism: Cellular and molecular phenotypes in pluripotent stem cell derived-neurons
    Article Snippet: Irradiated CF1 mouse embryonic fibroblasts (MEFs) were from Applied StemCell and incubated in MEF medium: DMEM with 10% heat inactivated-FBS, 1% GlutaMAX (Life Technologies), and 1% antibiotics. .. Generation, maintenance, and characterizations of iPSCs were performed as described elsewhere [ ], with specific modifications described in the . iPSCs and hESC were cultured on MEF feeders in iPSC medium (Knockout DMEM (Life Technologies), containing 20% Knockout SR (Life Technologies), 1% GlutaMAX, 0.1 mM β-mercaptoethanol (Life Technologies), 1% antibiotics, 1% NEAA, and 10 ng/ml basic fibroblast growth factor (bFGF) (Peprotech). ..

    Article Title: Demarcation of Stable Subpopulations within the Pluripotent hESC Compartment
    Article Snippet: Fractionation of hESC based on REX1Venus expression reveals a previously hidden hierarchy within the pluripotent compartment, comprising undifferentiated and differentiation primed cells, which lacks the metastability observed in murine ESCs .. Human ESC culture and differentiation Human ESC line H1 (WiCell) was grown on mitotically-inactivated MEFs in hESC media (Knockout DMEM supplemented with 15% Knockout SR, 1× Non Essential Amino Acids, 1× Glutamax, 1× 2ME (all Invitrogen) and 16 ng/ml bFGF (Peprotech) and passaged with Collagenase type IV (Invitrogen). .. For antibiotic selection experiments, cells were cultured in hESC media with or without the addition of 1.5 ug/ml puromycin.

    Article Title: Generation of neural progenitor cells by chemical cocktails and hypoxia
    Article Snippet: HUCs were collected and cultured in REGM (Lonza, CC-4127) as described , . .. Generation of ciNPCs For neural progenitor cell induction from MEFs and TTFs, initial cells cultured in DMEM for 24 h were transferred into KSR medium including knockout DMEM (Life Technologies, 10829-018), 15% knockout serum replacement (Life Technologies, 10828), 1% NEAA (Life Technologies, 35050), 1% GlutaMax (Life Technologies, 35050-061), 1% sodium pyruvate (Life Technologies, 11360), 0.1 mM β-mercaptoethanol (Life Technologies, 21985-023) and 1 000 U/ml leukemia inhibitory factor (LIF) (Chemicon, ESG1107). ..

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    Article Snippet: Next, ES-GFP cells were seeded onto MEFs and cultured under various experimental conditions. .. Standard culture was conducted in DMEM+LIF medium, consisting of Knockout DMEM (Life Technologies) supplemented with 15% ES-qualified FBS (Life Technologies), 0.1 mM nonessential amino acids (Sigma-Aldrich), 2 mM L-glutamine (Life Technologies), 0.1 mM β-mercaptoethanol (Sigma-Aldrich), 50 U/mL penicillin (Life Technologies), 50 μg/mL streptomycin (Life Technologies), and 500 U/mL leukaemia inhibitory factor (LIF; Chemicon). .. Next, ES-GFP cells were cultured in media commonly used for myoblast culture: DMEM+FBS medium (high-glucose DMEM supplemented with 10% FBS and antibiotics) or DMEM+HS (high-glucose DMEM supplemented with 2% horse serum [HS; Life Technologies] and antibiotics) or DMEM+FBS+HS (low-glucose DMEM [Life Technologies] supplemented with 20% FBS, 10% HS, 0.5% chicken embryo extract (Sera Laboratories), and antibiotics).

    Article Title: Zinc finger proteins orchestrate active gene silencing during embryonic stem cell differentiation
    Article Snippet: Embryonic stem cells were cultured and passaged on 0.1% gelatinized (Sigma-Aldrich, St. Louis, MO, USA) dishes, as reported previously ( ). .. E14 mouse ESCs were cultured in DMEM (Hyclone, Logan, Utah) or KNOCK-OUT™ DMEM (Gibco, Grand Island, NY, USA) supplemented with 15% FBS (Gibco, Grand Island, NY, USA), 2 mM l -glutamine, 55 μM β-mercaptoethanol, 1% (v/v) non-essential amino acid, 100 U/ml penicillin and 100 μg/ml streptomycin (all from Gibco, Grand Island, New York) and 500 U/ml ESGRO LIF (Millipore, Germany). ..

    Recombinant:

    Article Title: Aldehyde dehydrogenase 1 positive glioblastoma cells show brain tumor stem cell capacity
    Article Snippet: The human GBM cell lines LN18, LN229, LNZ308, and G139 were cultured in the Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS), 2 mM l -glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin (Invitrogen) under the standard cell culture conditions at 37°C and 5% CO2 . .. To investigate the neurosphere formation, the cells were grown in a serum-free neurobasal medium consisting of Knockout DMEM (Invitrogen), Nutrient Mixture Ham's F12 + l -glutamine (Invitrogen), and 10% Knockout-Serum Replacement (Invitrogen) with 1% N2-Supplement (Invitrogen), 1% Non-Essential Amino-Acids (Invitrogen), 0.1% natural mouse laminin (Invitrogen), 2 mM l -glutamine (Biochrom), 100 U/mL penicillin (Biochrom), 100 µg/mL streptomycin (Biochrom), 10 ng/mL basic fibroblast growth factor (bFGF; Invitrogen), and 50 ng/mL epidermal growth factor (EGF; human recombinant, Millipore). .. For nonadherent growth conditions, the culture dishes were covered with 0.1% gelatin type A (Sigma-Aldrich).

    Infection:

    Article Title: Vascular Smooth Muscle Cells Derived from Inbred Swine Induced Pluripotent Stem Cells for Vascular Tissue Engineering
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    Cell Culture:

    Article Title: Idiopathic autism: Cellular and molecular phenotypes in pluripotent stem cell derived-neurons
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    Article Title: Generation of neural progenitor cells by chemical cocktails and hypoxia
    Article Snippet: HUCs were collected and cultured in REGM (Lonza, CC-4127) as described , . .. Generation of ciNPCs For neural progenitor cell induction from MEFs and TTFs, initial cells cultured in DMEM for 24 h were transferred into KSR medium including knockout DMEM (Life Technologies, 10829-018), 15% knockout serum replacement (Life Technologies, 10828), 1% NEAA (Life Technologies, 35050), 1% GlutaMax (Life Technologies, 35050-061), 1% sodium pyruvate (Life Technologies, 11360), 0.1 mM β-mercaptoethanol (Life Technologies, 21985-023) and 1 000 U/ml leukemia inhibitory factor (LIF) (Chemicon, ESG1107). ..

    Article Title: Zinc finger proteins orchestrate active gene silencing during embryonic stem cell differentiation
    Article Snippet: Embryonic stem cells were cultured and passaged on 0.1% gelatinized (Sigma-Aldrich, St. Louis, MO, USA) dishes, as reported previously ( ). .. E14 mouse ESCs were cultured in DMEM (Hyclone, Logan, Utah) or KNOCK-OUT™ DMEM (Gibco, Grand Island, NY, USA) supplemented with 15% FBS (Gibco, Grand Island, NY, USA), 2 mM l -glutamine, 55 μM β-mercaptoethanol, 1% (v/v) non-essential amino acid, 100 U/ml penicillin and 100 μg/ml streptomycin (all from Gibco, Grand Island, New York) and 500 U/ml ESGRO LIF (Millipore, Germany). ..

    Stable Transfection:

    Article Title: Generation of liver-specific TGF-α/c-Myc-overexpressing porcine induced pluripotent stem-like cells and blastocyst formation using nuclear transfer
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    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, 2021-04
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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