The following matrices were assessed for the ability to support pluripotent growth and subsequent cardiac differentiation: 9 µg/cm2 growth-factor reduced Matrigel (1:200, Corning) in DMEM/F12; 625 ng/cm2 vitronectin peptide (Synthemax II-SC, 1:320, Corning) in ultrapure water (1:50 also tested); 1 µg/cm2 full length recombinant human vitronectin (1:50, Primorigen) in D-PBS with CaCl2 and MgCl2; 2.5 µg/cm2 laminin-521 (1:80, Biolamina) in DPBS; 2 µg/cm2 truncated recombinant human laminin-511 iMatrix-511 (1:50, Iwai North America, Foster City, CA, USA) in DPBS; 1 µg/cm2 rH E-cadherin (1:25, StemAdhere, Primorigen/Stemcell Technologies); and 10 µg/cm2 fibronectin (1:20, EMD Millipore) in D-PBS. All were used at 2 mL per well of a 6-well (9.6 cm2). Matrices were assessed on both 6-well polystyrene tissue culture plates and untreated plates (both from Greiner). Also tested were Synthemax-T 6-well plates, and fibronectin mimetic plates (both from Corning) and 10 µg/cm2 Pronectin (Sigma-Aldrich).
Synthemax 2 sc
Manufactured by Corning
Sourced in United States, Germany
Synthemax II-SC is a cell culture surface coating product developed by Corning. It is designed to enhance cell attachment and growth in various cell culture applications. The product provides a synthetic extracellular matrix-like environment to support cell proliferation and differentiation.
Automatically generated - may contain errors
Lab products found in correlation
Mtesr1 medium, by STEMCELL (2 mentions)
Fibronectin, by Merck Group (1 mentions)
Laminin 521, by BioLamina (1 mentions)
Tryple select enzyme, by Thermo Fisher Scientific (1 mentions)
Cellbind surface, by Corning (1 mentions)
Stemmacs msc expansion media kit xf, by Miltenyi Biotec (1 mentions)
Cellstart ctstm, by Thermo Fisher Scientific (1 mentions)
Neon transfection system, by Thermo Fisher Scientific (1 mentions)
Gentle cell dissociation reagent (gcdr), by STEMCELL (1 mentions)
Cloner, by STEMCELL (1 mentions)
Hincii, by New England Biolabs (1 mentions)
Rpmi b27, by Thermo Fisher Scientific (1 mentions)
Matrigel, by Corning (1 mentions)
Rpmi medium, by Thermo Fisher Scientific (1 mentions)
Iwr 1, by Merck Group (1 mentions)
Ascorbic acid, by Merck Group (1 mentions)
Activin a, by Tebu-Bio (1 mentions)
Df19 9 11t h, by WiCell (1 mentions)
Sodium bicarbonate, by Merck Group (1 mentions)
Hepes, by Thermo Fisher Scientific (1 mentions)
6 protocols using synthemax 2 sc
1
Pluripotent Stem Cell Cardiac Differentiation
2
Expansion of Chondrogenic Progenitor Cells
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Tissue fragments were placed in 115 cm2 tissue-culture flasks with reclosable lids (TPP, Switzerland) in the presence of StemMACS-MSC expansion Media kit XF (Miltenyi Biotec GmbH, Germany) after coating with CELLstartTM CTSTM (Gibco/Thermo Fisher Scientific) or Synthemax® II-SC (Corning) as adhesion substrates for “GMP grade I” and “GMP grade II” conditions, respectively (see Table 1 ). For the coating with CELLstartTM CTSTM, flasks were incubated with the reagent diluted 1:50 in DPBS for 2 h at 37°C. Regarding the coating with Synthemax® II-SC, flasks were incubated with the reagent diluted 1:40 in sterile water for 2 h at RT.
At confluence, CPC were harvested using TrypLETM Select Enzyme (Gibco/Thermo Fisher Scientific), then seeded at 8–10 × 104 cells/cm2 and expanded in T flasks (75–150 cm2), HYPERFlask® (1720 cm2) or HYPERStack®-12 (6000 cm2) culture vessels (Corning). Thanks to their negatively charged, highly hydrophilic CellBIND® surface (Corning), designed to facilitate cell attachment and spreading, HYPERFlasks and HYPERStacks did not require any coating.
For CPC MCB and PPCB generation (see below), the above indicated StemMACS-MSC expansion Media kit XF was substituted by its GMP counterpart, MSC-Brew GMP Medium (test lot kindly provided by Miltenyi Biotec GmbH).
CPC expanded in GMP grade conditions I and II are thereafter indicated as CPC-I and CPC-II, respectively.
At confluence, CPC were harvested using TrypLETM Select Enzyme (Gibco/Thermo Fisher Scientific), then seeded at 8–10 × 104 cells/cm2 and expanded in T flasks (75–150 cm2), HYPERFlask® (1720 cm2) or HYPERStack®-12 (6000 cm2) culture vessels (Corning). Thanks to their negatively charged, highly hydrophilic CellBIND® surface (Corning), designed to facilitate cell attachment and spreading, HYPERFlasks and HYPERStacks did not require any coating.
For CPC MCB and PPCB generation (see below), the above indicated StemMACS-MSC expansion Media kit XF was substituted by its GMP counterpart, MSC-Brew GMP Medium (test lot kindly provided by Miltenyi Biotec GmbH).
CPC expanded in GMP grade conditions I and II are thereafter indicated as CPC-I and CPC-II, respectively.
3
Precise CRISPR-Cas9 Editing of hiPSCs
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hiPSCs were dissociated with Gentle Cell Dissociation Reagent (STEMCELL Technologies), and 1 × 105 cells were transfected with the Alt-R Cas9 RNP complex and the ssODN (both IDT) using the Neon Transfection System (Invitrogen) at 1200 V/30 ms/1 pulse (for gRNA and ssODN sequence, see table S5). Cells were plated in a Synthemax II-SC (Corning)–coated plate using TeSR-E8 supplemented with CloneR (STEMCELL Technologies). After recovery, 1000 cells were plated in a Synthemax II-SC–coated 10-cm dish in TeSR-E8 supplemented with CloneR and refreshed daily. After ~14 days, single cell–derived hiPSC colonies were manually picked, and one-half was used for DNA isolation with QuickExtract Solution (Lucigen). Positive clones were identified by PCR screening of the region of interest, followed by successful digestion by restriction enzyme Hinc II (New England Biolabs), according to the manufacturer’s instructions. Primer sequences are shown in table S6. The introduced mutation was confirmed by Sanger sequencing, performed by the Leiden Genome Technology Center.
4
Cardiac Differentiation of Human Pluripotent Stem Cells
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In this study two hPSC lines were used namely, the hiPSC line DF19-9-11T.H (WiCell) and the hESC line (HES3 reporter line NKX2-5(eGFP/w)22 (link)) kindely provided by Dr. David Elliott. hPSCs were routinely expanded in Synthemax II-SC (corning) coated plates in mTESR1 medium (STEMCELL Technologies). Before cardiac differentiation induction, hPSCs were replated on Matrigel® (Corning) coated plates and cultured in mTESR1 medium until reaching 80%-90% confluency. Briefly, expansion medium was replaced by RPMI Medium (ThermoFisher Scientific) supplemented with B27 without insulin (RPMI/B27, ThermoFisher Scientific), 12 µM CHIR99021 (Biogen Cientifica SL), 80 ng/mL Activin A (Tebu-bio) and 50 µg/mL Ascorbic acid (Sigma-Aldrich). After 24 hr, the medium was completely replaced by RPMI/B27 supplemented with 5 µM IWR-1 (Sigma-Aldrich) and 50 µg/mL Ascorbic acid (Sigma-Aldrich). At day 3, i.e. 72 hr after day 0, medium was exchanged for RPMI/B27 supplemented with 5 µM IWR-1 until day 6. Medium was then exchanged every two days, until day 15. At day 15, cell preparations containing > 80% cardiac troponin T-positive CMs (confirmed by flow cytometry) were obtained.
5
Isotopic Labeling of hESCs in mTESR1
6
Maintenance and Differentiation of hiPSCs
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In this work, the hiPSC cell line iPS-DF6-9-9 T.B, purchased from WiCell Bank, was used. This cell line is vector free and was derived from foreskin fibroblasts with a karyotype 46, XY. Maintenance of hiPSC culture was performed using mTeSR1 medium (STEMCELL Technologies) in 6-well tissue culture plates coated with Matrigel (BD Biosciences) diluted 1 : 30 in DMEM/F12. Prior to differentiation, hiPSCs were adapted in the same xeno-free culture system used for differentiation by using TeSR2 medium (STEMCELL Technologies) or Essential 8 medium (Thermo Fisher Scientific). Plates were coated with Synthemax II-SC (Corning), a synthetic vitronectin-based peptide, at a concentration of 5 μg/cm2, Vitronectin XF (STEMCELL Technologies), a defined human recombinant protein, at a concentration of 1.25 μg/cm2 or laminin from murine origin (Sigma) at a concentration of 2.5 μg/cm2. Enzyme-free passaging was performed using EDTA (Thermo Fisher Scientific) solution diluted in PBS at a concentration of 0.5 mM. Cells were incubated for 5 min with EDTA at room temperature and flushed with culture medium. For cell counting, a sample of 100 μL was incubated in 400 μL of Accutase for 7 min at room temperature and samples were diluted in trypan blue. Phase contrast images were obtained using a Leica DMI 3000B microscope and a digital camera Nikon DXM 1200.
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