Methods for Enhanced Production of Cardiomyocytes from Embryonic Stem Cells

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Temporal modulation of p02gas affects differentiation. Fraction of cells that were cardiomyocytes, total number of cells, and total number of cardiomyocytes as determined by flow cytometry of trypsin-dispersed cell samples immunostained with MF-20 after 10 days of differentiation. Cells were started at either 142 or 7 mmHg and then switched to the other condition on day 2, 4, 6, 8, or not at all. The 10 different experimental p02gas histories are shown at the bottom of the Figure, and the day of change of the p02gas is noted. The number of independent experiments (n), each with three replicates, is given at the top of each column. Data are mean of the results of the independent experiments± s.d. where n = 3, or ± range where n = 2.Dependence of V max on p02gas at which cells were cultured. The maximal normoxic OCR (V max) of undifferentiated mES cells ( closed circles), day 3 EBs  (triangles), and day 10 tissue (open circles) is shown as a function of the culture p02gas· The fit of these data using Eqn. (3) is also shown. Data for V max with undifferentiated ES cells are reported as mean± s.d. for at least 4 independent experiments, and data for differentiating cells are from single experiments. Enhanced cardiomyogenesis in serum-free medium. Fraction of cells that were cardiomyocytes (top), total number of cells (middle), and total number of cardiomyocytes (bottom) for cells cultured at constant p02gas conditions of 7, 36, or 142 mmHg for 11 days. Cells were maintained in serum containing DMEM for all 11 days ( open bars) or were changed to a serum-free ITS medium after 5 days (solid bars). The number fraction of cardiomyocytes was determined by flow cytometry of trypsin-dispersed cell samples immunostained with MF-20. Data shown as mean± s.d. for 3 replicate wells in a single experiment.
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Inventors
Professor Clark Colton
Department of Chemical Engineering, MIT
External Link (mitei.mit.edu)
Daryl Powers
Department of Chemical Engineering, MIT
Jeffrey Millman
Department of Chemical Engineering, MIT
Amanda Diienno
Department of Chemical Engineering, MIT
Managed By
Michelle Hunt
MIT Technology Licensing Officer
Patent Protection

Methods and compositions for enhanced differentiation from embryonic stem cells

US Patent 9,029,147

Applications

This technology increases the number of cardiomyocytes produced using embryonic stem (ES) cells and may be used for the treatment of cardiovascular disease.

Problem Addressed

Cardiomyocytes generated from ES cells in vitro have successfully survived, fused with surrounding tissue, and been retained in animal hosts making them ideal candidates for clinical transplantation therapy. In order for these clinical applications to come to fruition, sufficient numbers of cardiomyocytes need to be produced. This invention describes methods to increase the production of cardiomyocytes from ES cells and create cell culture populations enriched in cardiomyocytes.  

Technology

ES cells exposed to low oxygen levels for an extended period of time differentiate into cardiomyocytes even in the absence of exogenously added growth factors and agents. First, cells are cultured at an oxygen partial pressure lower than atmospheric partial pressure over two to eight days. A second culture step may be further pursued in which the oxygen partial pressure is subsequently raised for another two to eight days. This simple method generates on average sixty cardiomyocytes from a single ES cell and allows for cell populations with a higher proportion of cardiomyocytes. 

Advantages

  • Increases the number of cardiomyocytes and cardiomyocyte progenitors
  • Simple method produces cardiomyocyte enriched populations without cardiomyocyte selection
  • No need for exogenous agents to promote differentiation