Application protocol

Differentiation, isolation, and analysis of cardiomyocytes derived from hPSCs

In this application protocol, we describe the differentiation of human pluripotent stem cells (hPSCs) into cardiomyocytes, the isolation of the resulting cardiomyocytes, and their analysis using flow cytometry or immunofluorescence microscopy.

Materials

The following is a listing of reagents, instruments, and consumables needed for each step of this protocol. These products are for research use only.

For hPSCs differentiation into cardiomyocytes

  • StemMACS™ iPS-Brew XF, human (# 130-104-368)
  • TrypLE™ Select Enzyme (1×), no phenol red, liquid (Life Technologies®)
  • Soybean Trypsin Inhibitor, powder (Life Technologies)
  • StemMACS Thiazovivin (# 130-104-461)
  • RPMI 1640 + L-glutamine (Life Technologies)
  • B-27® Supplement (Life Technologies)
  • B-27 Supplement, minus insulin (Life Technologies)
  • StemMACS CHIR99021 (# 130-103-926)
  • StemMACS IWR-1-edo (# 130-110-491)
  • 12-well plates coated with Matrigel® hESC-Qualified Matrix (Corning®)
  • Dulbecco’s phosphate-buffered saline (DPBS) without Ca²+ and Mg²+ (e.g., Lonza, #BE17-512F)

For cell harvest and preparation

  • Multi Tissue Dissociation Kit 3 (# 130-110-204)
  • MACS® SmartStrainers (70 μm) (# 130-098-462)
  • Cell culture medium with 20 % fetal bovine serum (FBS)
  • Phosphate-buffered saline (PBS), pH 7.4

For cardiomyocyte isolation from hPSCs

  • PSC-Derived Cardiomyocyte Isolation Kit, human (# 130-110-188)
  • PBE buffer: Prepare a solution containing phosphate-buffered saline (PBS), pH 7.2, 0.5 % bovine serum albumin (BSA), and 2 mM EDTA by diluting MACS BSA Stock Solution (# 130‑091-376) 1:20 with autoMACS® Rinsing Solution (# 130‑091-222). Keep buffer cold (2−8 °C). Degas buffer before use, as air bubbles could block the column.
    ▲ Note: EDTA can be replaced by other supplements such as anticoagulant citrate dextrose formula-A (ACD-A) or citrate phosphate dextrose (CPD). BSA can be replaced by other proteins such as human serum albumin, human serum, or fetal bovine serum (FBS). Buffers or media containing Ca2+ or Mg2+ are not recommended for use.
  • MACS Columns and MACS Separators: For optimal purity and recovery, the use of LS Columns for depletion of non-cardiomyocytes as well as for the subsequent positive selection of PSC-derived cardiomyocytes is strongly recommended. Positive selection and depletion can also be performed  using the autoMACS Pro Separator.
ColumnMax. number of labeled cellsMax. number of total cellsSeparator
Depletion or positive selection
LS5×10⁶5×10⁶MidiMACS™, QuadroMACS™,
VarioMACS, SuperMACS II
autoMACS1×1071×107autoMACS Pro
▲ Note: Column adapters are required to insert certain columns into the VarioMACS™ or SuperMACS™ II Separators. For details refer to the respective MACS Separator data sheet.

For flow cytometry or immunofluorescence microscopy analysis

For analysis by flow cytometry:

  • MACSQuant® Analyzer 10 (# 130-096-343)
  • Anti-Cardiac Troponin T-FITC, human, mouse, rat; clone REA400 (# 130-106-687)
  • Anti-α-Actinin (Sarcomeric)-VioBlue®, human, mouse, rat; clone REA402 (# 130-106-935)
  • Anti-Myosin Heavy Chain-APC, human, mouse, rat; clone REA399 (# 130-106-215)
  • Anti-MLC2a-APC, human, mouse, rat; clone REA398 (# 130-106-143)
  • Anti-MLC2v-PE, human, mouse, rat; clone REA401 (# 130-106-133)
  • Inside Stain Kit (# 130-090-477)
  • autoMACS Running Buffer – MACS Separation Buffer (# 130-091-221)
  • MACS BSA Stock Solution (# 130-091-376)
  • Dulbecco’s phosphate-buffered saline (DPBS)

For analysis by immunofluorescence microscopy:

  • Anti-α-Actinin (Sarcomeric) pure, human, mouse, rat (# 130‑112-755) or Anti-Cardiac Troponin T pure, human, mouse, rat (# 130-112-756)
  • Phosphate-buffered saline (PBS)
  • autoMACS Running Buffer (# 130-091-221)
  • Secondary antibody, e.g., Anti-IgG (H+L)-Vio® 515, human (# 130‑112‑760)
  • Inside Stain Kit (# 130-090-477) for the fixation and permeabilization of cells

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Protocol


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This protocol includes detailed instructions for preparing necessary reagents and materials and then carrying out each step.