Human disc tissue

Abstract

This invention provides disc stem cells, processes for obtaining and culturing disc stem cells, and methods for repairing damaged or diseased disc tissue comprising the use of the disc stem cells of the invention.

1 - 23 . (canceled) 
     
     
         24 . A method of amplifying a population of nucleus pulposus cells comprising the steps of:
 a. suspending isolated nucleus pulposus cells in a medium comprising approximately 14-15% serum, basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), and 30-40% human neonatal foreskin fibroblast (NFF) cell supernatant; and   b. plating the suspension onto gelatin-coated tissue culture plates for 5-14 days,   wherein said cells grow as an attached monolayer, thereby amplifying a nucleus pulposus cell population.   
     
     
         25 . The method of  claim 24 , wherein said nucleus pulposus cells are mammalian nucleus pulposus cells. 
     
     
         26 . The method of  claim 25 , wherein said nucleus pulposus cells are human nucleus pulposus cells. 
     
     
         27 . The method of  claim 24 , wherein said nucleus pulposus cells are isolated from degenerated disc tissue. 
     
     
         28 . The method of  claim 24 , wherein said nucleus pulposus cells are isolated from healthy disc tissue. 
     
     
         29 . The method of  claim 28 , wherein said healthy disc tissue is fetal, neonatal, or young adult tissue. 
     
     
         30 . The method of  claim 24 , wherein said FGF is present at a concentration of 10 ng/ml. 
     
     
         31 . The method of  claim 24 , wherein said EGF is present at a concentration of 10 ng/ml. 
     
     
         32 . The method of  claim 24 , wherein said medium is DMEM/F12. 
     
     
         33 . The method of  claim 24 , wherein said serum is fetal calf serum (FCS). 
     
     
         34 . The method of  claim 24 , wherein said nucleus pulposus cells reach confluency after a 5-7 day incubation. 
     
     
         35 . The method of  claim 24 , wherein said nucleus pulposus cells reach confluency after a 7-14 day incubation. 
     
     
         36 . The method of  claim 24 , wherein said nucleus pulposus cells are plated at a density of 50,000 cells/cm2. 
     
     
         37 . The method of  claim 24 , wherein at least a portion of said nucleus pulposus cells demonstrate notochordal cell morphology. 
     
     
         38 . The method of  claim 24 , further comprising the step of cryopreserving said enriched disc stem cell population. 
     
     
         39 - 55 . (canceled) 
     
     
         56 . A method of amplifying and enriching a disc stem cell population comprising the steps of:
 a. suspending a sphere-like cluster of disc stem cells or an isolated disc stem cell from said sphere-like cell cluster in a medium comprising 6-10% serum, basic fibroblast growth factor (bFGF), and epidermal growth factor (EGF) and lacking methylcellulose;   b. distributing the suspension onto ultra-low binding culture plates; and   c. culturing said suspension for 8-16 days,   wherein said cluster or cell grows into a large aggregate cluster of heterogeneous morphology, thereby amplifying and enriching a disc stem cell population.   
     
     
         57 . The method of  claim 56 , wherein said disc stem cell population is a mammalian disc stem cell population. 
     
     
         58 . The method of  claim 56 , wherein said disc stem cell population is a human disc stem cell population. 
     
     
         59 . The method of  claim 56 , wherein said disc stem cell population is isolated from degenerated disc tissue. 
     
     
         60 . The method of  claim 56 , wherein said disc stem cell population is isolated from healthy disc tissue. 
     
     
         61 . The method of  claim 60 , wherein said healthy disc tissue is fetal, neonatal, or young adult tissue. 
     
     
         62 . The method of  claim 56 , wherein said serum is present at a concentration of 10%. 
     
     
         63 . The method of  claim 56 , wherein said FGF is present at a concentration of 10 ng/ml. 
     
     
         64 . The method of  claim 56 , wherein said EGF is present at a concentration of 10 ng/ml. 
     
     
         65 . The method of  claim 56 , wherein said serum is fetal calf serum (FCS). 
     
     
         66 . The method of  claim 56 , wherein said medium is DMEM/F12, N10, or a combination thereof. 
     
     
         67 . The method of  claim 56 , wherein said medium further comprises putrescine, progesterone, sodium selenite, transferrin, and insulin. 
     
     
         68 . (canceled) 
     
     
         69 . The method of  claim 56 , wherein said suspension is distributed at a concentration of 1×10 5  cells/ml. 
     
     
         70 . The method of  claim 56 , further comprising the step of producing a single cell suspension from a sphere-like disc stem cell cluster using enzymatic digestion prior to step (a). 
     
     
         71 - 72 . (canceled) 
     
     
         73 . The method of  claim 56 , wherein at least a portion of said disc stem cell population expresses markers of mature disc cells after incubation with chondrogenic differentiating media. 
     
     
         74 . The method of  claim 73 , wherein said markers comprise collagen-2 alpha, vimentin, or a combination thereof. 
     
     
         75 . The method of  claim 56 , further comprising the step of cryopreserving said large cluster of heterogeneous morphology. 
     
     
         76 . (canceled) 
     
     
         77 . An amplified nucleus pulposus cell population obtained using the method of  claim 24 . 
     
     
         78 . A disc tissue derived clonal stem cell population obtained using the method of  claim 24 . 
     
     
         79 . A disc tissue derived heterogeneous stem cell population obtained using the method of  claim 56 . 
     
     
         80 - 97 . (canceled) 
     
     
         98 . A method of preventing, inhibiting, or decreasing the likelihood of damage to or disease of a cartilage-containing joint of a subject, comprising the step of administering to said subject the amplified disc stem cell population of  claim 24 , thereby preventing, inhibiting, or decreasing the likelihood of damage to or disease of the cartilage-containing joint of said subject. 
     
     
         99 . The method of  claim 98 , wherein the administration is percutaneous. 
     
     
         100 . The method of  claim 98 , wherein the administration is directly to the joint of said subject as part of a surgical procedure. 
     
     
         101 . The method of  claim 98 , wherein said joint is the hip joint. 
     
     
         102 . The method of  claim 98 , wherein said joint is the knee joint. 
     
     
         103 . The method of  claim 98 , wherein said joint is the spinal joint.