All experiments inducing human primordial germ cell-like cells (hPGCLCs) from hiPSCs were approved by the University of Pennsylvania IRB. hiPSCs were cultured on plates coated with Recombinant laminin-511 E8 (iMatrix-511 Silk, Nacalai USA) and were maintained under a feeder-free condition in the StemFit® Basic04 medium (Ajinomoto) containing basic FGF (Peprotech) at 37 °C under an atmosphere of 5% CO2 in air. Prior to passaging or the induction of differentiation, hiPSC cultures were treated with a 1:1 mixture of TrypLE Select (Life Technologies) and 0.5 mM EDTA/PBS for 14 min at 37 °C to dissociate them into single cells. 10 μM ROCK inhibitor (Y-27632; Tocris) was added in culture medium for 1 day after passaging hiPSCs.
Stemfit basic04 medium
Manufactured by Ajinomoto
Sourced in United States
StemFit Basic04 medium is a cell culture medium designed for the maintenance and expansion of human pluripotent stem cells. It provides the necessary nutrients and growth factors to support the growth and self-renewal of these cells under feeder-free and xeno-free conditions.
Automatically generated - may contain errors
Lab products found in correlation
Basic fgf, by Thermo Fisher Scientific (5 mentions)
Rock inhibitor y 27632, by Bio-Techne (5 mentions)
Tryple select, by Thermo Fisher Scientific (5 mentions)
Imatrix 511 silk, by Nacalai Tesque (1 mentions)
Evos xl core microscope, by Thermo Fisher Scientific (1 mentions)
Y 27632, by Biogems (1 mentions)
6 well plate, by Thermo Fisher Scientific (1 mentions)
Stemfit basic04ct, by Thermo Fisher Scientific (1 mentions)
Rneasy micro kit, by Qiagen (1 mentions)
Nextseq 500 instrument, by Illumina (1 mentions)
Nextera xt dna library preparation kit, by Illumina (1 mentions)
Smart seq ht kit, by Takara Bio (1 mentions)
6 protocols using stemfit basic04 medium
1
Inducing Human Primordial Germ Cells
2
Feeder-free hiPSC Culture Maintenance
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hiPSCs were cultured on plates coated with recombinant laminin-511 E8 (iMatrix-511 Silk, Nacalai USA) and maintained under feeder-free conditions in StemFit Basic04 medium (Ajinomoto) containing 20 ng/ml basic FGF (Peprotech) at 37°C under a 5% CO2 atmosphere. Before passaging or the induction of differentiation, hiPSC cultures were treated with a 1:1 mixture of TrypLE Select (Life Technologies) and 0.5 mM EDTA/PBS for 15 min at 37°C to dissociate them into single cells. Subsequently, 10 μM ROCK inhibitor (Y-27632; Tocris) was added in culture medium for 1 day after passaging hiPSCs.
3
Expansion of Induced Pluripotent Stem Cells
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Once iPSC colonies reached a sufficient size for picking, they were picked and transferred into fresh wells for expansion. An EVOS XL Core microscope (Life Technologies) was sterilised and placed into a cell culture hood under UV irradiation to ensure sterility. For each colony, 1 well of a 24-well plate was prepared by coating with laminin (recombinant iMatrix Laminin-511 silk E8) and pre-warming StemFit Basic04 medium (Ajinomoto) supplemented with a 10 µm ROCK inhibitor (Y-27632, Biogems, Westlake Village, CA, USA). Subsequently, the iPSC colony was identified under the microscope and scratched using a sterile 200 µL pipette tip to delineate its perimeter from neighbouring fibroblasts. If needed, a cross-hatch pattern was created to facilitate colony detachment. The detached colony fragments were aspirated with a 200 µL pipette and transferred into the prepared well with a pre-warmed medium. The medium was refreshed 24 h post-colony picking to remove the ROCK inhibitor.
4
Maintenance and Passaging of Human iPSCs
5
Generation of Induced Pluripotent Stem Cell-Derived Primordial Germ-Like Cells
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The iPSC (9A13 XY) line used in this study was established in a previous study [Hwang et al. [10 (link)]]. iPSCs were cultured on plates coated with recombinant laminin-511 E8 (BG iMatrix-511 Silk, Peprotech, Cranbury, NJ) and were maintained under feeder-free conditions in StemFit Basic04 medium (Ajinomoto, Tokyo, Japan) containing basic FGF (Peprotech) at 37°C under an atmosphere of 5% CO2 in air. For passaging or induction of differentiation, the cells were treated with a 1:1 mixture of TrypLE Select (Life Technologies, Waltham, MA) and 0.5 mM EDTA/PBS to enable their dissociation into single cells, and 10 mM ROCK inhibitor (Y-27632; Tocris, Abingdon, United Kingdom) was added.
PGCLCs were induced from iPSCs via iMeLCs as described previously [Sasaki et al. [4 (link)]] and purified using the surface markers EpCAM and INTEGRINα6. Total RNA was extracted from iPSCs and PGCLCs by using an RNeasy Micro Kit (Qiagen, Venlo, Netherlands) according to the manufacturer’s instructions. cDNA was synthesized using 1 ng of purified total RNA, and cDNA libraries were constructed for RNA sequencing by using a SMART-Seq HT Kit (Takara, Shiga, Japan) and a Nextera XT DNA Library Preparation Kit (Illumina, San Diego, CA) according to the manufacturers’ instructions. The libraries were sequenced using a single-end sequencing protocol on an Illumina NextSeq 500 instrument.
PGCLCs were induced from iPSCs via iMeLCs as described previously [Sasaki et al. [4 (link)]] and purified using the surface markers EpCAM and INTEGRINα6. Total RNA was extracted from iPSCs and PGCLCs by using an RNeasy Micro Kit (Qiagen, Venlo, Netherlands) according to the manufacturer’s instructions. cDNA was synthesized using 1 ng of purified total RNA, and cDNA libraries were constructed for RNA sequencing by using a SMART-Seq HT Kit (Takara, Shiga, Japan) and a Nextera XT DNA Library Preparation Kit (Illumina, San Diego, CA) according to the manufacturers’ instructions. The libraries were sequenced using a single-end sequencing protocol on an Illumina NextSeq 500 instrument.
6
Induction of Human Primordial Germ Cells
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The experiments on the induction of human primordial germ cell-like cells (hPGCLCs) from human induced pluripotent stem cells (hiPSCs) were approved by Institutional Review Board of University of Pennsylvania. hiPSCs were culture on a plate coated with Recombinant laminin-511 E8 (iMatrix-511 Silk, Nacalai USA) and were maintained under a feeder-free condition in the StemFit® Basic04 medium (Ajinomoto) containing basic FGF (Peprotech) at 37 ºC under an atmosphere of 5% CO2 in air. For the passage or the induction of differentiation, the cells were treated with a 1 to 1 mixture of TrypLE Select (Life Technologies) and 0.5 mM EDTA/PBS for 14 min at 37 ºC to enable their dissociation into single cells. 10 M ROCK inhibitor (Y-27632; Tocris) was added in culture medium for 1 day after passaging hiPSCs.
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