Stem Cell Biology

Cardiac Stem Cell Protocols

Cardiomyocyte Differentiation
Enrichment of Cardiomyocyte

Cardiomyocyte Differentiation
Cardiomyocytes are readily identifiable since, within 1 to 4 days after plating, they spontaneously contract. With continued differentiation, the amount of spontaneous beating foci increases and all the EBs may include localized beating cells. Fully differentiated cardiomyocytes often stop contracting, but can be maintained in culture for many weeks. Several chemicals have proven helpful for the enhancement of cardiogenic differentiation of ES cells. They are retinoic acid (R2625), 0.5 to 1.5% dimethylsulfoxide (DMSO) (D2438), and 5-aza-dC (A3656).



Enrichment of Cardiomyocyte
To use hES cell-derived cardiomyocytes in therapeutic applications, it will be benefical to produce a population of cells highly enriched for cardiomyocytes.

  • Wash the differentiated cultures containing beating cardiomyocytes three times with PBS (P7059) or a low calcium solution. The low calcium solution contains 120 mM NaCl (S8776), 5.4 mM KCl (P9327), 5 mM MgSO4 (M3409), 5 mM sodium pyruvate (P5280), 20 mM glucose (G8644), 20 mM taurine (T0625), and 10 mM HEPES (H6147) at pH 6.9.
  • Discard the PBS. Add an appropriate amount of collagenase (C2799) to cover the cells. The 1 mg/ml collagenase (C2799) is prepared in the low calcium solution supplemented with 30 mM CaCl2 (C8106).
  • Incubate at 37° for 1 to 2 hours.
  • Resuspend the cells in a high potassium solution. The high potassium solution contains 85 mM KCl (P9327), 30 mM K2HPO4 (P2222), 5 mM MgSO4 (M3409), 1 mM EGTA (E4378), 2 mM Na2ATP (A6419), 5 mM sodium pyruvate (P5280), 5 mM creatin (C0780), 20 mM taurine (T0625), and 20 mM glucose (G8644) at pH 7.2.
  • Incubate at 37 °C for 15 minutes for more complete dissociation. Gently pipette to achieve a uniform cell suspension.
  • Transfer the cells suspension into a 10-ml conical tube. Spin at 1200 rpm for 5 minutes.
  • Aspirate the supernatant. Add 3 ml high glucose DMEM (D5796, D5671, D6429, D6546, D1145, D6171, D0422, D5648, D1152, D7777) containing 20% FBS (F2442, F6178) and resuspend the cells by gentle pipetting.
  • Prepare a Percoll (P4937) gradient as described. 1. Mix Percoll (P4937) with 8.5% NaCl (S8776) (9:1) to reach a physiological osmotic equilibrium. 2. Dilute the Percoll-8.5% NaCl solution with 8.5% NaCl to a final Percoll concentration of 40.5 and 58.5%, which correspond to a physical density of 1.065 and 1.069 g/ml, respectively.
  • Add 3 ml of 58.5% Percoll (P4937) to the bottom of a 10-ml conical tube and then gently add 3 ml of 40.5% Percoll on top of the 58.5% Percoll.
  • Add 3 ml of cell suspension onto the top of the Percoll solution by using a pipette leaning against the inner wall of the tube. Be sure to do it very gently so as not to disturb the Percoll layers.
  • Centrifuge at 1500 rpm for 30 minutes.
  • After centrifuging, two layers of cells will be observed; one on top of the Percoll (fraction I) and a layer of cells at the interface of the two layers of Percoll (fraction III). Cells can also be found in the 40.5% Percoll layer (Fraction II) and the 58.5% Percoll layer (fraction IV).

 

Generally, 20 to 40% of cells in fraction III and 50 to 70% of cells in fraction IV express cardiac-specific tropin I (cTnI), a subunit of the tropin complex that provides a calcium-sensitive molecular switch for the regulation of striated muscle contraction.

  • Carefully aspirate the cells in fraction III and cells in fraction IV.
  • Wash the cell fractions twice with PBS (P7059).

 

Protocols taken in part from:

  • Yang, X. et al. Methods in Enzymology 2006, 418, Elsevier Inc.

 

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