Fibronectin fragment to be used for stem cell production
Inventors
Otsuji, Tomomi • Nishie, Toshikazu • KATO, Risa • Okamoto, Sachiko • Enoki, Tatsuji • Mineno, Junichi
Assignees
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Publication Number
US-11999972-B2
Publication Date
2024-06-04
Expiration Date
Abstract
Stem cells can be efficiently proliferated by culturing the stem cells in the presence of a novel recombinant fibronectin fragment.
Core Innovation
The invention provides recombinant polypeptides comprising, in the same molecule, three sets of human fibronectin III repeats: human fibronectin III-1 to 7 repeats, human fibronectin III-8 to 10 repeats, and human fibronectin III-12 to 14 repeats. For each repeat set, the polypeptide includes specific amino acid sequences defined by SEQ ID NOs corresponding to the repeat sets, and variants having at least 90% identity to the respective SEQ ID NO amino acid sequences.
The recombinant polypeptide is defined to retain a function of maintaining an undifferentiated state of stem cells while having a molecular weight of 100 kDa or less. The described constructs include recombinant fibronectin fragment polypeptides exemplified as FCH-296 and DCH-296, which correspond to the specific repeat compositions and variant definitions.
The invention further describes using the recombinant fibronectin fragment polypeptides to culture stem cells, including induced pluripotent stem cells and neural stem cells, in formats where the recombinant polypeptide is provided as a coating or solid phase. Comparative results are described showing adhesion, long-term culture performance, marker retention for undifferentiated and pluripotent markers, and outcomes for single-cell cloning, including performance versus full-length fibronectin and existing fibronectin fragments, and compatibility with xeno-free media.
Claims Coverage
Independent claim coverage centers on a recombinant polypeptide with a specific fibronectin III repeat composition, defined sequence and identity requirements, an undifferentiation-maintaining function, and a molecular-weight limit. Dependent claims narrow the repeat subsets, add specific sequence IDs, and extend the scope to coated solid-phase formats and cell-culture devices or carriers.
Specific fibronectin III repeat composition with sequence and 90% identity variants
A recombinant polypeptide comprising, in the same molecule, three repeats selected from human fibronectin III-1 to 7 repeats with sequences of SEQ ID NO: 1 to 7 or variants having at least 90% identity, human fibronectin III-8 to 10 repeats comprising sequences of SEQ ID NO: 8, 9, 10 or variants having at least 90% identity, and human fibronectin III-12 to 14 repeats comprising sequences of SEQ ID NO: 12, 13, 14 or variants having at least 90% identity.
Undifferentiation-maintaining function for stem cells
The recombinant polypeptide retains a function of maintaining an undifferentiated state of stem cells.
Molecular weight limited to 100 kDa or less
The recombinant polypeptide has a molecular weight of 100 kDa or less.
Repeat subset narrowing for the III-1 to 7 portion
The recombinant polypeptide variant specifies three repeats comprising selected subsets of human fibronectin III-1 to 7 repeats, using the corresponding SEQ ID NO amino acid sequences or variants having at least 90% identity.
Specific sequence IDs for the recombinant polypeptide
The recombinant polypeptide comprises an amino acid sequence corresponding to SEQ ID NO: 19 or 20, or an amino acid sequence having at least 90% identity to SEQ ID NO: 19 or 20, and has a molecular weight of 100 kDa or less.
Coated solid phase using the recombinant polypeptide
A solid phase is coated with the recombinant polypeptide.
Cell-culture device or carrier with coated recombinant polypeptide
A solid phase serves as a device or carrier for cell culture, where the carrier is coated with a recombinant polypeptide.
Specific solid-phase formats coated with the recombinant polypeptide
The coated solid phase is selected from a dish, plate, flask, bag, bead, membrane, or slide glass, each coated with a recombinant polypeptide.
Coverage is anchored by a recombinant polypeptide defined by a specific human fibronectin III repeat composition, sequence or 90% identity variants, a requirement to maintain an undifferentiated state of stem cells, and a molecular weight of 100 kDa or less. Dependent claims then narrow repeat subsets and add specific sequence IDs, and they further extend to coated solid phases and enumerated cell-culture formats.
Stated Advantages
Provides efficient proliferation of stem cells while maintaining undifferentiated or pluripotent markers.
Shows higher or equivalent performance compared with full-length fibronectin and existing fibronectin fragments.
Supports adhesion, long-term culture performance, and marker retention.
Enables single-cell cloning with higher or equivalent performance.
Compatible with xeno-free media.
Documented Applications
Culturing stem cells, including induced pluripotent stem cells and neural stem cells, using the recombinant fibronectin fragment polypeptides.
Coating or solid-phase presentation of the recombinant fibronectin fragment polypeptides to culture cells on cell culture devices and carriers.
Single-cell cloning of stem cells on formats involving the coated recombinant polypeptide.
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