# US 12,213,997 B1 · Compositions and methods for supporting nucleus pulposus (NP) cell phenotype and biosynthesis > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 12,213,997 B1 - Document ID: US12213997B1 - Status: Granted - Kind: B1 - Application number: 17/165,011 - Filed: 2021-02-02 - Granted: 2025-02-04 - Earliest claimed: 2020-02-04 - Assignees: Washington University - Applicants: Lori Setton; Marcos Barcellona; Julie Speer - Inventors: Lori Setton; Marcos Barcellona; Julie Speer - Web page: https://patentgrep.com/patent/US12213997B1 ## CPC - A61K 35/32: Bones; Osteocytes; Osteoblasts; Tendons; Tenocytes; Teeth; Odontoblasts; Cartilage; Chondrocytes; Synovial membrane - A61K 35/545: Embryonic stem cells; Pluripotent stem cells; Induced pluripotent stem cells; Uncharacterised stem cells - A61K 47/60: the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol - C07K 14/78: Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG] - C12N 5/0068: {General culture methods using substrates (for specific animal cell type C12N5/06)} - C12N 5/0654: {Osteocytes, Osteoblasts, Odontocytes; Bones, Teeth} - C12N 2533/30: Synthetic polymers - C12N 2533/52: Fibronectin; Laminin - A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D) - C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00) - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) ## Abstract Among the various aspects of the present disclosure is the provision of compositions and methods for the generation of a supporting matrix for cells, such as stem cells and NP cells (and precursors thereof). For example, an NP cell-specific phenotype precursor cell can be an adult NP cell, such as a cell taken from the adult and degenerated intervertebral disc and/or a progenitor cell, such as stem cells, induced pluripotent cells, pluripotent cells, precursor cells, or stromal cells. ## Claims ### Claim 1 A biocompatible composition comprising: a PEG maleimide biocompatible polymer component; and at least one cell-adhesive peptide, wherein the cell-adhesive peptide is between about 5 and about 30 amino acids long; the cell-adhesive peptide comprises at least one syndecan-binding or integrin-binding peptide selected from SEQ ID NO: 1, CGG-terminated SEQ ID NO: 1, SEQ ID NO: 2, and C-terminated SEQ ID NO: 2; the cell-adhesive peptide is coupled to the biocompatible polymer component to form a cell-adhesive peptide-functionalized monomer; the cell-adhesive peptide-functionalized monomer is crosslinked to form a cell-adhesive peptide-functionalized polymer, wherein the cell-adhesive peptide-functionalized polymer has a density greater than 10% (w/v); the biocompatible composition has a stiffness of about 10 kPa; and the biocompatible composition comprises a cell-adhesive peptide density between about 50 μM and about 100 μM sufficient to support nucleus pulposus (NP) cell-specific morphology, biosynthesis, or phenotype. ### Claim 2 (depends on 1) The biocompatible composition of claim 1, wherein the cell-adhesive peptide is derived from a sequence found in naturally occurring laminin proteins, a functional portion of the LG domains of full length laminin, isoforms thereof, or a functional fragment or variant thereof. ### Claim 3 (depends on 1) The biocompatible composition of claim 1, wherein the cell-adhesive peptide is a laminin-mimetic peptide. ### Claim 4 (depends on 3) The biocompatible composition of claim 3, wherein the laminin-mimetic peptide is a laminin-based integrin-binding peptide or a laminin-based syndecan-binding peptide. ### Claim 5 (depends on 1) The biocompatible composition of claim 1, comprising a first integrin-binding peptide and a second integrin-binding peptide. ### Claim 6 (depends on 1) The biocompatible composition of claim 1, wherein the biocompatible polymer component comprises a terminal component capable of conjugating a cell-adhesive peptide. ### Claim 7 (depends on 6) The biocompatible composition of claim 6, wherein the terminal component comprises maleimide, cysteine, or carboxylate. ### Claim 8 (depends on 1) The biocompatible composition of claim 1, wherein the cell-adhesive peptide-functionalized monomer is crosslinked via coupling of the biocompatible polymer component via side group coupling, a terminal group on the biocompatible polymer component, a sulfo-, a dithiol crosslinker, a PEG crosslinker, an amine, or EDC/NHS coupling. ### Claim 9 (depends on 8) The biocompatible composition of claim 8, wherein the dithiol crosslinker is SH-PEG-SH. ### Claim 10 (depends on 1) The biocompatible composition of claim 1, wherein if the cell-adhesive peptide density is increased, the biocompatible composition stiffness increases. ### Claim 11 (depends on 1) The biocompatible composition of claim 1, wherein the cell-adhesive peptide comprises a terminal group capable of conjugating to a biocompatible polymer component comprising a bioconjugation component capable of conjugating with the terminal group. ### Claim 12 (depends on 1) The biocompatible composition of claim 1, wherein the cell-adhesive peptide comprises a thiol terminal group capable of conjugating to a biocompatible polymer component comprising maleimide. ### Claim 13 (depends on 1) The biocompatible composition of claim 1, wherein the at least one cell-adhesive peptide is a combination of a syndecan-binding peptide and an integrin-binding peptide. ### Claim 14 (depends on 1) The biocompatible composition of claim 1, wherein the at least one cell-adhesive peptide is at least two of integrin-binding, syndecan-binding, or cadherin-binding peptides. ### Claim 15 (depends on 3) The biocompatible composition of claim 3, wherein the laminin-mimetic peptide further comprises a sequence selected from the group consisting of PPFLMLLKGSTR (SEQ ID NO: 3), EGYGEGYIGSR (SEQ ID NO: 4), AGQWHRVSRWG (SEQ ID NO: 5), KQNCLSSRASFRGCVRNLRLSR (SEQ ID NO: 6), and combinations thereof. ### Claim 16 (depends on 1) The biocompatible composition of claim 1, wherein the cell-adhesive peptide-functionalized polymer density is between about 15% (w/v) and about 40% (w/v). ### Claim 17 (depends on 1) The biocompatible composition of claim 1, further comprising a population of undifferentiated precursor cells, a population of stem cells, a population of nucleus pulposus (NP) cell precursors, a population of adult primary NP cells, or a population of primary intervertebral disc (IVD) cells, or a combination thereof. ### Claim 18 (depends on 16) The biocompatible composition of claim 16, wherein the NP cell precursors are NP progenitor cells. ### Claim 19 (depends on 1) The biocompatible composition of claim 1, wherein the biocompatible composition comprises a cell having an NP cell-specific phenotype characterized by the gene expression of COL1A1, ACAN, COL2A1, GLUT1, or CDH2, or combinations thereof. ### Claim 20 (depends on 1) The biocompatible composition of claim 1, wherein the biocompatible composition comprises a population of NP cell-specific phenotype precursor cells selected from IVD cells, primary adult NP cells, NP precursor cells, adult NP cells, or pathological NP cells, or combinations thereof. ### Claim 21 (depends on 16) The biocompatible composition of claim 16, wherein the NP cell precursors are undifferentiated precursor cells. ### Claim 22 (depends on 1) The biocompatible composition of claim 1, further comprising at least one progenitor cell in its undifferentiated state. ### Claim 23 (depends on 1) The biocompatible composition of claim 1, wherein the biocompatible composition further comprises stem cells selected from adipose stromal/stem cells (ASCs), adipose-derived stem cells (ADSCs), amniotic fluid stem cells, bone marrow-derived mesenchymal stem/stromal cells (BMSCs), bone marrow stem cells, cord blood stem cells, embryonic stem (ES) cells, hematopoietic stem cells, induced pluripotent stem cells (iPSCs), mesenchymal stromal, non-embryonic (adult) stem cells, pluripotent stem cells (PSCs), progenitor cells, induced pluripotent stem (iPS) cells), induced pluripotent stem cells (iPSCs), mesenchymal stromal cells, mesenchymal stem cells (MSCs), cord blood stem cells, umbilical cord-derived progenitor cells, umbilical cord-MSCs, umbilical cord-derived progenitor cells, amniotic fluid stem cells, and other progenitor cells. ### Claim 24 (depends on 23) The biocompatible composition of claim 23, wherein the stem cells promote cell attachment, promote the secretion of inflammatory mediating factors, or promote the secretion of matrix degrading protease mediating factors. ### Claim 25 (depends on 24) The biocompatible composition of claim 24, wherein the inflammatory mediating factors are selected from interleukin (IL)-1 receptor agonist (RA), SIL-6R, soluble tumor necrosis factor receptor (sTNFR) I, sTNFRII, tissue inhibitor of metalloproteinases (TIMP) 1, TIMP 2, TIMP 3, TIMP 4, and combinations thereof. ### Claim 26 (depends on 1) The biocompatible composition of claim 1, wherein the biocompatible composition supports progenitor cell secretions of chemokines that exert an anti-inflammatory effect upon neighboring cells. ### Claim 27 (depends on 1) The biocompatible composition of claim 1, further comprising progenitor cells cultured in or on the biocompatible composition, wherein the progenitor cells secrete inflammatory-mediating factors that result in inflammatory mediating effects upon neighboring cells. ### Claim 28 (depends on 27) The biocompatible composition of claim 27, wherein the inflammatory-mediating factors are selected from IL-1RA, sIL-6R, sTNFRI, STNFRII, and combinations thereof. ### Claim 29 (depends on 1) The biocompatible composition of claim 1, further comprising progenitor cells cultured in or on the biocompatible composition, wherein the progenitor cells secrete protease mediating factors which result in protease mediating effects upon neighboring cells. ### Claim 30 (depends on 29) The biocompatible composition of claim 29, wherein the protease mediating factors are selected from TIMP_1, TIMP 2, TIMP_3, TIMP_4, and combinations thereof. ### Claim 31 (depends on 1) The biocompatible composition of claim 1, wherein the biocompatible composition supports progenitor cells capable of secreting extracellular matrix biosynthesis promoting chemokines within neighboring cells. ### Claim 32 (depends on 31) The biocompatible composition of claim 31, wherein anabolic effects are achieved through secretion of extracellular matrix proteins. ### Claim 33 (depends on 1) The biocompatible composition of claim 1, wherein the biocompatible composition is suitable for use as an injectable material mixed with cells or cell-free, wherein the biocompatible composition has a viscosity that does not exceed G′ of 100 Pa and G″ of 25 Pa at temperatures between about 4° C. and about 37° C. ### Claim 34 (depends on 1) The biocompatible composition of claim 1, wherein the biocompatible composition is suitable for delivery to a defect site when injected in its precursor form mixed with cells or cell-free through a 12 G-22 G needle. ### Claim 35 (depends on 1) The biocompatible composition of claim 1, wherein the biocompatible composition is a 3D cell supporting matrix or coats a tissue culture surface. ### Claim 36 (depends on 1) The biocompatible composition of claim 1, wherein the biocompatible composition is capable of being used in cell-polymer culture constructs in vitro in volumes between about 10 mL and about 1 mL and between about 0 million cells/mL and up to about 10 million cells/mL. ### Claim 37 (depends on 1) The biocompatible composition of claim 1, wherein the biocompatible composition delivers cells to an intervertebral disc. ### Claim 38 (depends on 1) The biocompatible composition of claim 1, wherein the cell-adhesive peptide densities and co-combinations of coupling to peptide-functionalized polymer components modulate a range of cell phenotypic changes. ### Claim 39 (depends on 1) The biocompatible composition of claim 1, wherein the biocompatible composition supports cell survival, when introduced to a population of cells, in a peptide-functionalized polymer component solution, and crosslinked into a three-dimensional construct. ### Claim 40 (depends on 1) The biocompatible composition of claim 1, wherein the biocompatible composition is capable of delivering and localizing NP precursor cells or primary human NP cells and peptide-functionalized polymer components to defects in an intervertebral disc in a subject. ### Claim 41 (depends on 1) A method of supporting adult human NP cells towards a healthy nucleus pulposus (NP) cell-specific phenotype or delivering undifferentiated progenitor cells comprising administering to a subject the biocompatible composition of claim 1. ### Claim 42 (depends on 41) The method of claim 41, wherein the subject has intervertebral disc degeneration or damage.