Method for purifying cardiomyocytes or programmed cardiomyocytes derived from stem cells or fetuses
Inventors
Hattori, Fumiyuki • Fukuda, Keiichi
Assignees
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Publication Number
US-9115342-B2
Publication Date
2015-08-25
Expiration Date
Abstract
An object of the present invention is to develop a method for purify cardiomyocytes at a high degree of purification and at a high yield from a cell mixture comprising cardiomyocytes derived from fetuses and stem cells using various features which have not been previously expected to be used for purification of cardiomyocytes or which are newly found, wherein said method is carried out without undergoing any genetic modification or without adding any special proteins or biologically active agents.
Core Innovation
The invention relates to a method of selecting cardiomyocytes and/or Brachyury+/Nkx2.5-cells capable of differentiating into cardiomyocytes from a cell mixture containing cardiomyocytes, Brachyury+/Nkx2.5-cells, and non-cardiomyocyte cells. The method is performed by culturing the cell mixture in a culture medium under a sugar-free condition or a condition wherein sugar content is 111.20 μM or less.
In addition to the sugar-free condition, the method selects the cardiomyocytes and/or Brachyury+/Nkx2.5-cells by applying one or more selected conditions chosen from low calcium, low nutritional, lactic acid-supplemented, aspartic acid/glutamic acid-supplemented, and pyruvic acid-supplemented conditions. The starting cell mixture is derived from totipotent cells, pluripotent cells, or stem cell having similar characteristics to those of embryonic stem cell through differentiation induction.
The stem cells having similar characteristics to those of embryonic stem cells are characterized by surface markers specific for embryonic stem cells, embryonic stem cell specific gene expression, and/or an ability to form a teratoma. The claimed selection approach is based on culturing the mixed cells under defined metabolic and stress-like culture conditions to preferentially select cardiomyocytes and/or Brachyury+/Nkx2.5-cells capable of differentiating into cardiomyocytes.
Claims Coverage
The independent claim is directed to selecting cardiomyocytes and/or Brachyury+/Nkx2.5-cells from a mixed cell population by culturing under a defined sugar-free or very low sugar condition together with one or more additional specified culture conditions. The independent claim contains three core inventive features, while the dependents further refine defined parameter ranges and optional dead-cell elimination treatments.
Sugar-free or very low sugar culture medium selection
Culturing the cell mixture in a culture medium under a sugar-free condition or a condition wherein sugar content is 111.20 μM or less.
Additional selection conditions for selecting cardiomyocytes and/or Brachyury+/Nkx2.5-cells
Culturing the cell mixture under one or more conditions selected from the group consisting of a low calcium condition, a low nutritional condition, a lactic acid-supplemented condition, an aspartic acid/glutamic acid-supplemented condition, and a pyruvic acid-supplemented condition to select the cardiomyocytes and/or Brachyury+/Nkx2.5-cells from the cell mixture.
Starting cell mixture derived from totipotent/pluripotent/embryonic-stem-like stem cells via differentiation induction
Using a cell mixture derived from totipotent cells, pluripotent cells, or stem cell having similar characteristics to those of embryonic stem cell through differentiation induction, wherein the stem cells having similar characteristics to those of embryonic stem cells have surface markers specific for embryonic stem cells, have embryonic stem cell specific gene expression, and/or have an ability to form a teratoma.
Overall, the independent claim covers selecting cardiomyocytes and/or differentiating Brachyury+/Nkx2.5-cells from a mixed population by combining a sugar-free or 111.20 μM or less sugar culture condition with one or more additional specified culture conditions, using a starting mixture derived from totipotent, pluripotent, or embryonic-stem-like stem cells characterized by embryonic stem cell markers, gene expression, and/or teratoma formation ability.
Stated Advantages
Not explicitly described in patent.
Documented Applications
Not explicitly described in patent.
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