Embryonic stem cell generation and culture

2.1.3 Embryonic stem cell generation and culture

2.1.3.1 Mouse embryonic stem cells

The mouse CM-selectable embryonic stem cell (mESC) line (αMHC-neoR “A6-line”;

R1 background) was generated by electroporation of a plasmid encoding for a neomycin resistance gene (neoR), under the control of the cardiomyocyte restricted α-myosin heavy chain (αMHC) promoter (Rogge, Dissertation 2007), as described previously (Klug et al., 1996).

2.1.3.1.1 Culture of mouse embryonic stem cells

Mouse ESCs were cultured on mitotically inactive MEFs (25,500 cells/cm²; Section 2.1.2.1.3) in ESC culture medium (see appendix A2). Medium was exchanged every day. Once mESC-colonies reached 80-90 % confluency, cells were detached (Section 2.1.2.1.5) with 0.25 % trypsin-EDTA and split at a 1:5 ratio. mESC-colonies were seeded onto new MEF feeder layer plates in ESC medium. Undifferentiated mESCs were cultured for at least 3 passages, prior to the initiation of cardiac differentiation.

2.1.3.1.2 Scale up differentiation of mouse αMHC-NeoR ESCs

The differentiation of mESCs was scaled up into spinner flasks, as described previously (Christalla, Dissertation 2010; Niebruegge et al., 2008). Undifferentiated mESCs were dissociated into single cells with trypsin (Section 2.1.2.1.5). Cells were resuspended in differentiation medium (see appendix A2) and pre-plated for 40 mins to minimize the MEF content. Cell suspensions were subsequently counted with a Neubauer cell counting chamber. Spinner flask culture vessels (125 ml) equipped with a bulb-shaped glass stirrer (Techne, F7988) were inoculated with 50 ml of medium

2. Materials and Methods

5% CO2 incubator. Flasks were filled up to 100 ml differentiation medium after 24 hrs, followed by half medium exchange every 48 hrs. CM selection at day 11 was initiated by the addition of 400 µg/ml Geneticin (G418, PAA) for a further 6 days, in order to eliminate non-myocytes.

2.1.3.1.3 Digestion of embryoid bodies into single cardiomyocytes

At culture day 17, beating embryoid bodies (EBs) were harvested and dissociated into single CMs. EBs were transferred into two clean 50 ml polypropylene tubes. The tubes were left to rest for approximately 5-10 mins to allow the EBs to settle to the bottom of the tubes. The supernatant was very carefully removed and EBs were washed with pre-warmed 1X PBS and pelleted at 300 x g for 5 mins at RT. The supernatant again was carefully removed and EBs were resuspended in 6 ml Collagenase type I (Sigma-Aldrich, C0130; see appendix A2), containing DNase I (20 µg/ml; Calbiochem). The EBs were triturated and then incubated at 37 °C for 60–90 mins with agitation on a mechanical rocking platform (Biometra) to dissociate the EBs and yield single cells. Once all the EBs were dissociated, each tube was filled with pre-warmed 1X PBS and pelleted at 500 x g for 5 mins at RT. Cells were then resuspended in pre-warmed 6 ml 0.25% Trypsin-ETDA and were gently triturated for 5-10 mins until all cell clumps were dissociated. The enzymatic digestion was inactivated with 24 ml differentiation medium + DNase I (20 µl/ml). The cell suspensions were then passed through a pre-wetted 70 µm cell strainer (BD falcon) into a fresh 50 ml polypropylene tube. The cells were pelleted at 200 x g for 5 mins at 4 °C, resuspended in 10 ml differentiation medium and the tubes were pooled together. Cells were counted with (1:1) 0.4% trypan blue solution using a Neubauer cell counting chamber to determine the number and viability of cells. Cells were subsequently used for experiments, with a small cell fraction (~1 x 10⁶ cells) fixed in 70% ethanol (EtOH) to assess the purity of CM via flow cytometry (FC) analysis (Section 2.1.4).

2. Materials and Methods

2.1.3.2 Human embryonic stem cells

Experimentation with human embryonic stem cells were approved by the Robert-Koch-Institute (www.rki.de; approval #12 from 13.09.2005 to W.H. Zimmermann according to §11 Stammzellgesetz) and the Human Embryonic stem cell (hES2) line was obtained from the lab of Prof. Gordon Keller, Toronto, Canada. hES2 line contained a targeted red fluorescence protein (tdRFP) reporter gene to the human ROSA26 locus (Irion et al., 2007). hES2 cells were initially adapted to culture with hESC medium (see appendix A2) on γ-irradiated hFFBs, prior to cardiac differentiation (Soong et al., 2012; Zimmermann et al., 2015). hESC-medium was exchanged daily until colonies covered 80% of the culture flasks.

2.1.3.2.1 hES2 2D cardiac differentiation

Cardiac differentiation in 2D culture was performed on hES2 cells using an optimized protocol (Hudson et al., 2012). All 2D differentiation experiments were performed in 24 well plates filled with 0.5 ml culture medium and cultivated at 37°C in a 5% CO₂ incubator. Briefly, hES2 cells were plated at 5 x 10⁴ cells/cm² on Matrigel (growth factor reduced)-coated plates (1:60 diluted in 1X PBS; BD Biosciences, 354320) and cultured in 1:1 with hES-medium and irradiated hFFB-conditioned medium (conditioned for 2 days) with 10 ng/ml FGF2 (seeding phase). After 24 hrs seeding, hES2 cells were rinsed with RPMI medium (see appendix A2) and subsequently cultured in Mesoderm Induction medium (see appendix A2) for 3 days, followed by Cardiac Specification medium (see appendix A2) for 8 days, and thereafter Maturation medium for 1 day (see appendix A2). Differentiated cells were then cultivated for 5 days in Selection medium (0.28 ml/cm²; see appendix A2) to enrich the CM populations and eliminate non-myocytes via metabolic selection (Tohyama et al., 2013). Cells were then cultivated for a further 7 days in Maturation medium.

2. Materials and Methods

2.1.3.2.2 Single cell dissociation of hES2-cardiomyocyte monolayers

Enriched CM populations derived from hES2 cardiac differentiation in monolayer culture (Section 2.1.3.2.1) were dissociated into single cells. Cells were washed twice in 1X PBS. Next, cells were incubated with Accutase solution (See appendix A2; 0.1 ml/cm²) for 4 mins at RT. Cells were then incubated for a further 10-15 mins at 37 °C, until cells were detached from culture flasks and dissociated into single cells. The digestion procedure was deactivated with the addition of a threefold amount of serum-free CM medium containing 5 µmol/l Rock inhibitor (see appendix A2). Cells were triturated to aid in single cell dissociation, transferred into fresh polypropylene tubes and counted using the CASY counter (Section 2.1.2.3.5) to determine cell number and viability. Cells were subsequently used for experiments, with a small cell fraction (~1 x 10⁶ cells) fixed in 70% ethanol (EtOH) to assess the purity of CMs via Flow cytometry analysis (Section 2.1.4).