Frozen Stocks of CTRL and catKO cells at passage 35 were thawed on MEFs in conventional KSR/FGF2 media as above. At passage 37, the media was replaced with the defined media StemFit Basic03 (Ajinomoto, Cat# 34770). At passage 38, the cells were passaged onto recombinant Laminin iMatrix-511silk E8 (rLN511E8) (Fisher, cat # 502041396) in StemFit Basic03 (Ajinomoto, Cat# 34770). Cells were passaged as single cells using TrypLE (Fisher Scientific, Cat# 50591419) with 20,000 cells plated per well of a 6-well plate. Analysis of hPGCLC competency from cells cultured in feeder free-defined conditions was performed from passage 48 with 100ng/mL of Stem Cell Factor (SCF) in the PGCLC differentiation media (R&D Systems, Cat# 255-SC-010).
Stemfit basic03
Manufactured by Ajinomoto
Sourced in Japan
StemFit Basic03 is a culture medium designed for the maintenance and expansion of human pluripotent stem cells. It provides the necessary components to support the growth and self-renewal of these cells in vitro.
Automatically generated - may contain errors
Lab products found in correlation
Sb431542, by Fujifilm (3 mentions)
Chir99021, by Axon Medchem (3 mentions)
Imatrix 511, by Nippi (3 mentions)
Stemfit ak03n, by Ajinomoto (3 mentions)
Y 27632, by Fujifilm (2 mentions)
Ak03n, by Ajinomoto (2 mentions)
Fibroblast growth factor 2 (fgf2), by Fujifilm (2 mentions)
Countess 2 fl, by Thermo Fisher Scientific (1 mentions)
Chir99021, by Fujifilm (1 mentions)
Hbm msc, by PromoCell (1 mentions)
Sb431542, by Merck Group (1 mentions)
Sb431542, by R&D Systems (1 mentions)
Epidermal growth factor (egf), by R&D Systems (1 mentions)
Stem cell factor (scf), by R&D Systems (1 mentions)
Tryple, by Thermo Fisher Scientific (1 mentions)
Primesurface, by Sumitomo Bakelite (1 mentions)
Human serum albumin (hsa), by Fujifilm (1 mentions)
Fibronectin, by Merck Group (1 mentions)
Penicillin and streptomycin, by Thermo Fisher Scientific (1 mentions)
Recombinant human fgf2, by Fujifilm (1 mentions)
6 protocols using stemfit basic03
1
Feeder-free hPGCLC Differentiation
2
Directed neural crest induction from iPSCs
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Human iPSCs were seeded onto iMatrix-511-coated plates or dishes at a density of 3.6 × 103 cells/cm2 in StemFit AK03N medium and maintained in culture for 4 days. For NCC induction, the cells were cultured in StemFit Basic03 (equivalent to AK03N without bFGF, Ajinomoto, Tokyo, Japan) with 10 μM SB431542 (FUJIFILM Wako) and 1 μM CHIR99021 (Axon Medchem, Reston, VA, USA) for 10 days. Cell numbers were counted using a Countess II FL (Thermo Fisher Scientific). The medium was changed every 2 days from day 0 to 6 and every day from day 7 to 10.
3
Xeno-free induction of iMSCs from iPSCs
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Human iPSCs (1231A3) were cultured on an iMatrix-511 (Nippi, Tokyo, Japan)-coated cell culture plate in StemFit AK03N (Ajinomoto, Tokyo, Japan) as described previously42 . The xeno-free method to induce human iMSCs from human iPSCs is described elsewhere [D. Kamiya, personal communication]. Briefly, Cells were cultured in Stemfit Basic03 (Ajinomoto), which is comparable to AK03N minus FGF2, supplemented with SB431542 (Sigma, St. Louis, MO, USA) and CHIR99021 (Wako, Osaka, Japan), to induce NCCs. CD271+ cells were then sorted by FACS and expanded onto fibronectin-coated plates in Stemfit Basic03 supplemented with SB431542, EGF (R&D, Minneapolis, USA), and FGF2. iMSCs were induced and maintained with PRIME-XV MSC Expansion XSFM medium (Fujifilm Irvine Scientific, Tokyo, Japan). The cells were diluted at a ratio of 1:3 when they reached 70–80% confluence. Cells at passage 3 were used for the experiments. Human bone marrow mesenchymal stem cell (hBM-MSC) was obtained from PromoCell (Heidelberg, Germany) and cultured in PRIME-XV MSC Expansion XSFM medium.
4
3D Aggregation of hiPSCs for Tissue Engineering
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For 3-dimensional cell aggregates experiment, hiPSCs were dissociated into cell suspensions and seeded into 96 well U-bottom ultra-low attachment plates (PrimeSurface®, Sumitomo Bakelite, MS-9096U) in StemFit Basic03 (equivalent to AK03N without bFGF, Ajinomoto) in the presence of ROCK inhibitor (Y-27632, Wako, 034-24024). After aggregates with clear edges were formed, the culture medium was replaced with DMEM/F12 supplemented with 0.1% human serum albumin (HSA, Wako, 014-21543) and 10 ng bFGF (R&D, 236-EG). Aggregate cross-sectional area sizes were measured using Fiji ImageJ software (Schindelin et al., 2012 (link)). 1231A3 hiPSCs were used in all experiments unless otherwise stated.
5
iPSC-Derived Neural Crest and Mesenchymal Cells
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The induction and maintenance of iPSC-derived neural crest cells (iNCCs) and MSCs (iMSCs) were conducted as previously described, with minor modifications [16 (link), 17 (link)]. Briefly, iPSCs (1231A3) [27 (link)] were maintained in StemFit AK03N (Ajinomoto, Japan) supplemented with 10 µM Y27632 (Wako Pure Chemical, Japan) in dishes pre-coated with iMatrix-511 (nippi, Japan). iNCCs were induced in StemFit Basic03 (equivalent to AK03N without basic fibroblast growth factor (FGF2), Ajinomoto, Tokyo, Japan) with 10 µM SB-431542 (Selleck Chemicals, USA) and 1 µM CHIR99021 (Axon Medchem, Netherlands) for 10 d. iNCCs were maintained in StemFit Basic03 with 10 µM SB-431542 and 20 ng/mL recombinant human FGF2 (WAKO) and recombinant human EGF (R&D Systems, USA) onto plates pre-coated with fibronectin (Merck, USA). iMSCs were induced and maintained in PRIME-XV MSC Expansion XSFM (Fujifilm Irvine Scientific, USA) onto the fibronectin-coated plates. FOP-iMSCs and resFOP-iMSCs were generated as previously described [11 (link), 16 (link)] and cultured in αMEM (Invitrogen) supplemented with 5 ng/ml FGF2 (WAKO), 10% fetal bovine serum (FBS; Nichirei, Inc., Japan), and 0.5% penicillin and streptomycin (Invitrogen). The reagents are listed in Additional file 1 : Table S1.
6
Derivation of iMSCs from iPSCs
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iMSCs were derived from the 1231A3 strain of iPSCs (reprogrammed using episomal vectors, kindly provided from Yamanaka laboratory). The iMSCs were induced using a previously described method.10 (link) Briefly, iPSCs were cultured on an iMatrix-511 (Nippi, Tokyo, Japan)-coated cell culture dish containing StemFit AK03N (Ajinomoto, Tokyo, Japan) for 4 d and then cultured in StemFit Basic03 (equivalent to AK03N without basic fibroblast growth factor, Ajinomoto) containing 10 μM SB431542 (Fujifilm Wako, Osaka, Japan) and 1 μM CHIR99021 (Axon Medchem, Reston, VA, USA) for 10 d to induce neural crest cell (NCC) differentiation. NCCs were selectively harvested using a cell sorter and CD271 as a marker, and the sorted cells were seeded into human fibronectin (Thermo Fisher Scientific, Waltham, MA, USA)-coated dish containing Basic03 supplemented with 10 μM SB431542, 20 ng/mL epidermal growth factor (Fujifilm Wako), and fibroblast growth factor 2 (Fujifilm Wako) and expanded. Subsequently, differentiation into iMSCs was induced using PRIME-XV MSC Expansion XSFM medium (Fujifilm Wako). BM-MSCs were purchased from Lonza (Lot: 19TL168853, 19TL191055; Basel, Switzerland).
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