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US 11,685,903 B2Granted
Protein-induced pluripotent cell technology and uses thereof
AssigneeWayne State University
InventorsJianjun Wang, Qianqian Li
Life of this patent
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Abstract
A method of generating protein-induced pluripotent stem cells by delivering bacterially expressed reprogramming proteins into nuclei of starting somatic cells using the QQ-protein transduction technique, repeating several cell reprogramming cycles for creating reprogrammed protein-induced pluripotent stem cells, moving the reprogrammed cells into a feeder-free medium for expansion, and expanding and passaging the reprogrammed cells in a whole dish for generating homogeneous piPS cells. Also provided are the piPCS cells formed using this method and uses thereof.
Claims14
A method of generating a homogenous population of mammalian protein-induced pluripotent stem cells (piPSC), comprising:
- culturing mammalian somatic cells in the presence of a combination of QQ-reagent modified reprogramming proteins comprising QQ-reagent modified Sox2 and QQ-reagent modified Oct4, for at least a first or more reprogramming cycles, wherein the QQ-reagent modified Sox2 and QQ-reagent modified Oct4 are denatured or non-denatured recombinant proteins, wherein each reprogramming cycle comprises culturing mammalian cells with the combination of QQ-reagent modified reprogramming proteins for a period of about 3 hours to 5 days, wherein the combination of QQ-reagent modified reprogramming proteins comprises a QQ reagent, wherein the QQ reagent comprises polyethylenimine (PEI) and DOTAP/DOPE, wherein the QQ-reagent modified Sox2 and the QQ-reagent modified Oct4 are each present in the combination in a concentration of greater than 2 nanograms/milliliter (ng/ml) and no greater than 500 ng/ml, and wherein the cultured mammalian cells are passaged in a whole dish;
- and
- passaging the cultured mammalian cells in a whole dish under a feeder-free condition, with the proviso that colony picking is not performed, to generate a homogenous population of mammalian protein induced pluripotent stem cells.
A method of generating mammalian adult stem cells comprising:
- providing a homogenous population of mammalian protein-induced pluripotent stem cells (piPSC) by a method including:
- culturing mammalian somatic cells in the presence of a combination of QQ-reagent modified reprogramming proteins comprising QQ-reagent modified Sox2 and QQ-reagent modified Oct4, for at least a first or more reprogramming cycles, wherein the QQ-reagent modified Sox2 and QQ-reagent modified Oct4 are denatured or non-denatured recombinant proteins, wherein each reprogramming cycle comprises culturing mammalian cells with the combination of QQ-reagent modified reprogramming proteins for a period of about 3 hours to 5 days, wherein the combination of QQ-reagent modified reprogramming proteins comprises a QQ reagent, wherein the QQ reagent comprises polyethylenimine (PEI) and DOTAP/DOPE, wherein the QQ-reagent modified is Sox2 and the QQ-reagent modified Oct4 are each present in the combination in a concentration of greater than 2 nanograms/milliliter (ng/ml) and no greater than 500 ng/ml, wherein the cultured mammalian cells are passaged in a whole dish;
- and passaging the cultured mammalian cells in a whole dish under a feeder-free condition, with the proviso that colony picking is not performed, thereby generating a homogenous population of mammalian piPSC;
- and
- differentiating the piPSC to produce adult stem cells.
2 more elements
A method of generating mammalian adult progenitor cells comprising:
- providing a homogenous population of mammalian protein-induced pluripotent stem cells (piPSC) by a method including:
- culturing mammalian somatic cells in the presence of a combination of QQ-reagent modified reprogramming proteins comprising QQ-reagent modified Sox2 and QQ-reagent modified Oct4, for at least a first or more reprogramming cycles, wherein the QQ-reagent modified Sox2 and QQ-reagent modified Oct4 are denatured or non-denatured recombinant proteins, wherein each reprogramming cycle comprises culturing mammalian cells with the combination of QQ-reagent modified reprogramming proteins for a period of about 3 hours to 5 days, wherein the combination of QQ-reagent modified reprogramming proteins comprises a QQ reagent, wherein the QQ reagent comprises polyethylenimine (PEI) and DOTAP/DOPE, wherein the QQ-reagent modified is Sox2 and the QQ-reagent modified Oct4 are each present in the combination in a concentration of greater than 2 nanograms/milliliter (ng/ml) and no greater than 500 ng/ml, wherein the cultured mammalian cells are passaged in a whole dish;
- and passaging the cultured mammalian cells in a whole dish under a feeder-free condition, with the proviso that colony picking is not performed, thereby generating a homogenous population of mammalian piPSC;
- and
- differentiating the piPSC to produce adult progenitor cells.
2 more elements
A method of trans-differentiation to generate somatic cells comprising:
- providing a homogenous population of mammalian protein-induced pluripotent stem cells (piPSC) by a method including:
- culturing mammalian somatic cells in the presence of a combination of QQ-reagent modified reprogramming proteins comprising QQ-reagent modified Sox2 and QQ-reagent modified Oct4, for at least a first or more reprogramming cycles, wherein the QQ-reagent modified Sox2 and QQ-reagent modified Oct4 are denatured or non-denatured recombinant proteins, wherein each reprogramming cycle comprises culturing mammalian cells with the combination of QQ-reagent modified reprogramming proteins for a period of about 3 hours to 5 days, wherein the combination of QQ-reagent modified reprogramming proteins comprises a QQ reagent, wherein the QQ reagent comprises polyethylenimine (PEI) and DOTAP/DOPE, wherein the QQ-reagent modified is Sox2 and the QQ-reagent modified Oct4 are each present in the combination in a concentration of greater than 2 nanograms/milliliter (ng/ml) and no greater than 500 ng/ml, wherein the cultured mammalian cells are passaged in a whole dish;
- and passaging the cultured mammalian cells in a whole dish under a feeder-free condition, with the proviso that colony picking is not performed, thereby generating a homogenous population of mammalian piPSC;
- and
- differentiating the piPSC to produce terminally differentiated somatic cells.
2 more elements
Dependencies are inferred from the claim text.