METHODS, SYSTEMS, FACTORS, AND MEDIA FOR REDUCTION OF CELLULAR STRESS AND REACTIVE OXYGEN SPECIES
The present invention relates to methods, systems, factors, and media for the reduction of cellular stress and reduction of the amount reactive oxygen species. Embodiments of the invention reduce cellular stress by treating cells with a variety of molecules, such as certain inhibitors. Some embodiments may reduce the amount of reactive oxygen species in cell media through the use of scavengers.
1 . A method of mediating cellular stress during transition of a cell into another cell type, comprising:
culturing said cell under conditions that lead to said transition of said cell while carrying out two or more of the following:
reducing the available oxygen in a medium surrounding the cell;
treating the cell with an antioxidant;
inhibiting a cellular stress response pathway of the cell;
inhibiting an innate immune response of the cell; and
activating a cellular pathway that reduces the concentration of intracellular reactive oxygen species.
2 . A method of mediating cellular stress in a human blood precursor cell during transition of said precursor cell into a blood cell, comprising:
culturing said human blood precursor cell under conditions that lead to said transition of said precursor cell into a blood cell while carrying out one or more of the following:
reducing the available oxygen in a medium surrounding the human blood precursor cell;
treating the human blood precursor cell with an antioxidant;
inhibiting a cellular stress response pathway of the human blood precursor cell;
inhibiting an innate immune response of the human blood precursor cell; and
activating a cellular pathway that reduces the concentration of intracellular reactive oxygen species.
3 . The method of claim 1 or claim 2 , wherein said transition comprises differentiation of the cell to a more committed cell type.
4 . The method of claim 1 or claim 2 , wherein said transition comprises a conversion of the cell into an induced pluripotent stem cell.
5 . The method of claim 1 or claim 2 , wherein activating a cellular pathway that reduces the concentration of intracellular reactive oxygen species comprises treating the cell with a cAMP signaling activator.
6 . The method of claim 5 , wherein the cAMP signaling activator comprises Forskolin or IBMX.
7 . The method of claim 1 or claim 2 , wherein reducing the available oxygen in a medium surrounding the cell comprises placing the cell in a hypoxic environment.
8 . The method of claim 1 or claim 2 , wherein the antioxidant comprises a component selected from the group consisting of ascorbic acid, citric acid, vitamin E, selenium, melatonin, NAC, glutathione, thioredoxin, nicotinamide adenine dinucleotide phosphate, Superoxide dismutase, Catalase, and Glutathione peroxidase, and Citric acid monohydrate.
9 . The method of claim 8 , wherein the concentration of ascorbic acid is about 0.011-0.55 mg/ml.
10 . The method of claim 8 , wherein the concentration of citric acid is about 0.115-1.15 mg/ml.
11 . The method of claim 1 or claim 2 , wherein inhibiting the cellular stress response pathway comprises inhibiting mitochondrial p53 mediated apoptosis.
12 . The method of claim 1 or claim 2 , wherein inhibiting the cellular stress response pathway comprises inhibiting p38 mediated senescence.
13 . The method of claim 10 , wherein inhibition of the p38 mediated senescence comprises treating the cell with LY2228820 at a concentration range of about 20-500 nM.
14 . The method of claim 1 or claim 2 , wherein inhibiting the cellular stress response pathway comprises inhibiting endoplasmic reticulum stress.
15 . The method of claim 1 or claim 2 , wherein inhibiting the cellular stress response pathway comprises inhibiting non-mitochondrial calpain mediated stress.
16 . The method of claim 1 or claim 2 , wherein inhibiting the innate immune response comprises inhibiting myeloperoxidase production with a myeloperoxidase inhibitor.
17 . The method of claim 16 , wherein the myeloperoxidase inhibitor comprises 4-ABAH at a concentration range of about 100 μM.
18 . The method of claim 11 , wherein inhibiting the cellular stress response pathway comprises inhibiting mitochondrial p53 mediated apoptosis with pfilthrin-μ.
19 . The method of claim 1 or claim 2 , wherein inhibiting the cellular stress response pathway comprises inhibiting non-mitochondrial calpain mediated apoptosis of the cell.
20 . The method of claim 19 , wherein inhibiting non-mitochondrial calpain mediated apoptosis of the cell comprises treating the cell with MDL28170 at a concentration range of about 0.5-25 μM.
21 . The method of claim 1 or claim 2 , wherein reducing the available oxygen for a cell comprises reducing the amount of oxygen in a culture system to about 4%.
22 . The method of claim 1 or claim 2 , wherein the cell is an induced pluripotent stem (iPS) cell.
23 . A medium for the de novo generation of human blood cells, comprising two or more of the following:
an antioxidant;
an inhibitor of mitochondrial p53 mediated apoptosis;
an inhibitor of non-mitochondrial calpain mediated apoptosis;
an inhibitor of endoplasmic reticulum stress;
an innate immune response inhibitor;
an inhibitor of p38 mediated senescence; and
a cAMP signaling pathway activator.