Methods and compositions for producing germ cells from bone marrow derived germline stem cells

Abstract

The present invention relates to the use of bone marrow derived germline stem cells and their progenitors, methods of isolation thereof, and methods of use thereof.

1 . An isolated bone marrow cell that is mitotically competent, has an XX kayrotype and expresses Vasa, Oct-4, Dazl, Stella, Fragilis and optionally, Nobox, c-Kit and Sca-1.  
   
   
       2 . The isolated cell of  claim 1 , wherein the cell can produce oocytes after a duration of at least 1 week, 1 to about 2 weeks, about 2 to about 3 weeks, about 3 to about 4 weeks or more than about 5 weeks post transplantation into a host.  
   
   
       3 . The isolated cell of  claim 1 , wherein the cell can produce oocytes after a duration of less than 1 week post transplantation into a host.  
   
   
       4 . The isolated cell of  claim 1 , wherein the cell can produce oocytes after a duration of less than about 24 to about 48 hours post transplantation into a host.  
   
   
       5 . The isolated cell of  claim 2 , wherein the cell is a bone marrow derived female germline stem cell.  
   
   
       6 . The isolated cell of  claim 3 , wherein the cell is a bone marrow derived female germline stem cell progenitor.  
   
   
       7 . The isolated cell of  claim 1 , wherein the cell is a mammalian cell.  
   
   
       8 . The isolated cell of  claim 1 , wherein the cell is a human cell.  
   
   
       9 . The isolated cell of  claim 1 , wherein the cell is a non-embryonic cell.  
   
   
       10 . The isolated cell of  claim 1 , wherein the cell expresses Nobox.  
   
   
       11 . The isolated cell of  claim 1 , wherein the cell expresses c-Kit.  
   
   
       12 . The isolated cell of  claim 1 , wherein the cell expresses Sca-1.  
   
   
       13 . A method of in vitro fertilization of a female subject, said method comprising the steps of: 
 a) producing an oocyte by culturing the isolated cell of  claim 1  in the presence of an agent that differentiates the cell into an oocyte;    b) fertilizing the oocyte in vitro to form a zygote; and    c) implanting the zygote into the uterus of a female subject.    
   
   
       14 . A method of oocyte production, comprising culturing the isolated cell of  claim 1  in the presence of an agent that differentiates the cell into an oocyte, thereby producing an oocyte.  
   
   
       15 . The method of  claim 14 , wherein the agent is selected from the group consisting of a transforming growth factor, bone morphogenic protein, Wnt family protein, kit-ligand, leukemia inhibitory factor, meiosis-activating sterol, modulator of Id protein function and modulator of Snail/Slug transcription factor function.  
   
   
       16 . A pharmaceutical composition comprising a purified population of cells that are mitotically competent, have an XX karyotype and express Vasa, Oct-4, Dazl, Stella, Fragilis and optionally, Nobox, c-Kit and Sca-1 and a pharmaceutically acceptable carrier.  
   
   
       17 . The pharmaceutical composition of  claim 16 , wherein the cells are purified from the bone marrow.  
   
   
       18 . The pharmaceutical composition of  claim 16 , wherein the cells are mammalian cells.  
   
   
       19 . The pharmaceutical composition of  claim 16 , wherein the cells are human cells.  
   
   
       20 . The pharmaceutical composition of  claim 16 , wherein the purified population of cells is about 50 to about 55%, about 55 to about 60%, about 65 to about 760%, about 70 to about 75%, about 75 to about 80%, about 80 to about 85%, about 85 to about 90%, about 90 to about 95% or about 95 to about 100% of the cells in the composition.  
   
   
       21 . A method of oocyte production in a subject, comprising providing the pharmaceutical composition of  claim 16  to a tissue of the subject, wherein the cells engraft into the tissue and differentiate into oocytes, thereby producing oocytes in the subject.  
   
   
       22 . The method of  claim 21 , wherein the tissue is ovarian tissue.  
   
   
       23 . A method of inducing folliculogenesis in a subject, comprising providing the pharmaceutical composition of  claim 16  to the subject, wherein the cells engraft into a tissue of the subject and differentiate into oocytes within follicles, thereby inducing folliculogenesis in the subject.  
   
   
       24 . The method of  claim 23 , wherein the tissue is ovarian tissue.  
   
   
       25 . A method of treating infertility in a female subject in need thereof comprising administering a therapeutically effective amount of the pharmaceutical composition of  claim 16  to the subject, wherein the cells engraft into the ovary and differentiate into oocytes, thereby treating infertility.  
   
   
       26 . A method of repairing damaged ovarian tissue in a subject, comprising providing a therapeutically effective amount of the pharmaceutical composition of  claim 16  to the tissue, wherein the cells engraft into the tissue and differentiate into oocytes, thereby repairing the damaged tissue in the subject.  
   
   
       27 . The method of  claim 26 , wherein the damage is a result of exposure to chemotherapeutic drugs or radiation.  
   
   
       28 . The method of  claim 27 , wherein the chemotherapeutic drug is selected from the group consisting of busulfan, cyclophosphamide, 5-FU, vinblastine, actinomycin D, etoposide, cisplatin, methotrexate, and doxorubicin.  
   
   
       29 . The method of  claim 26 , wherein the damage is a result of a cancer, polycystic ovary disease, genetic disorder, immune disorder or metabolic disorder.  
   
   
       30 . A method of restoring ovarian function in a menopausal female subject, comprising administering a therapeutically effective amount of the pharmaceutical composition of  claim 16  to the subject, wherein the cells engraft into the ovary and differentiate into oocytes, thereby restoring ovarian function in the subject.  
   
   
       31 . The method of  claim 30 , wherein the menopausal female subject is in a stage of either peri- or post-menopause.  
   
   
       32 . A method for oocyte production in a subject, comprising contacting bone marrow derived female germline stem cells, or their progenitor cells, of the subject with an agent that differentiates the bone marrow derived female germline stem cells, or their progenitor cells, into oocytes, thereby producing oocytes in the subject.  
   
   
       33 . The method of  claim 32 , wherein the agent is selected from the group consisting of a transforming growth factor, bone morphogenic protein, Wnt family protein, kit-ligand, leukemia inhibitory factor, meiosis-activating sterol, modulator of Id protein function and modulator of Snail/Slug transcription factor function.  
   
   
       34 . A kit for oocyte production comprising an agent of  claim 33  and instructions for using the agent to differentiate the bone marrow derived female germline stem cells, or their progenitor cells, into oocytes, thereby producing oocytes.  
   
   
       35 . A method of expanding bone marrow derived female germline stem cells, or their progenitor cells, in vivo, ex vivo or in vitro, comprising contacting the bone marrow derived female germline stem cells, or their progenitor cells, with an agent that increases the amount of bone marrow derived female germline stem cells, or their progenitor cells, thereby expanding the bone marrow derived female germline stem cells, or their progenitor cells.  
   
   
       36 . The method of  claim 35 , wherein the amount of bone marrow derived female germline stem cells, or their progenitor cells, is increased with an agent that promotes cell proliferation or survival.  
   
   
       37 . The method of  claim 36 , wherein the agent is selected from the group consisting of an insulin-like growth factor, transforming growth factor, bone morphogenic protein, Wnt protein, fibroblast growth factor, sphingosine-1-phosphate, retinoic acid, inhibitor of glycogen synthase kinase-3, Bax inhibitor, caspase inhibitor, inhibitor of nitric oxide production and inhibitor of histone deacetylase activity.  
   
   
       38 . A kit for expanding bone marrow derived female germline stem cells, or their progenitor cells, comprising an agent of  claim 37 , and instructions for using the agent to increase the amount of bone marrow derived female germline stem cells or their progenitor cells, thereby expanding the bone marrow derived female germline stem cells, or their progenitor cells.  
   
   
       39 . A method for oocyte production in a subject, comprising contacting bone marrow derived female germline stem cells, or their progenitor cells, of the subject with an agent that increases the amount of bone marrow derived female germline stem cells, or their progenitor cells, thereby producing oocytes in the subject.  
   
   
       40 . The method of  claim 39 , wherein the agent increases the survival or proliferation of the cells, thereby increasing the amount of the cells.  
   
   
       41 . The method of  claim 40 , wherein the agent that increases survival or proliferation of the cells is selected from the group consisting of an insulin-like growth factor, transforming growth factor, bone morphogenic protein, Wnt protein, fibroblast growth factor, sphingosine-1phosphate, retinoic acid, inhibitor of glycogen synthase kinase-3, Bax inhibitor, caspase inhibitor, inhibitor of nitric oxide production and inhibitor of histone deacetylase activity.  
   
   
       42 . A method of restoring fertility to a female subject who desires restored fertility, comprising administering a therapeutically effective amount of bone marrow derived female germline stem cells, or their progenitor cells, to the subject, wherein the cells engraft into a tissue and differentiate into oocytes, thereby restoring fertility in the subject.  
   
   
       43 . The method of  claim 42 , wherein the tissue is ovarian tissue.  
   
   
       44 . A method of protecting fertility in a female subject undergoing or expected to undergo chemotherapy, radiotherapy or both treatments, comprising providing an agent that protects against reproductive injury prior to or concurrently with chemotherapy, radiotherapy or both treatments and providing a bone marrow derived female germline stem cell, or its progenitor cell, to the subject, wherein the cell engrafts into a tissue and differentiates into an oocyte, thereby protecting fertility in the subject.  
   
   
       45 . The method of  claim 44 , wherein the agent is selected from the group consisting of S1P, a Bax antagonist, or any agent that increases SDF-1 activity.  
   
   
       46 . A kit for protecting fertility in a female subject undergoing or expected to undergo chemotherapy, radiotherapy or both treatments, comprising an agent of  claim 45  and instructions for using the agent to protect bone marrow derived female germline stem cells, or their progenitor cells, against reproductive injury thereby protecting fertility in the female subject.  
   
   
       47 . A method for in vitro fertilization of a female subject, said method comprising the steps of: 
 a) producing an oocyte by contacting a bone marrow derived female germline stem cell, or its progenitor cell, with an agent that differentiates the bone marrow derived female germline stem cell, or its progenitor cell, into an oocyte;    b) fertilizing the oocyte in vitro to form a zygote; and    c) implanting the zygote into the uterus of a female subject.    
   
   
       48 . An isolated bone marrow cell that is mitotically competent, has an XY kayrotype and expresses Vasa and Dazl.  
   
   
       49 . The isolated cell of  claim 48 , wherein the cell is a mammalian cell.  
   
   
       50 . The isolated cell of  claim 48 , wherein the cell is a human cell.  
   
   
       51 . The isolated cell of  claim 48 , wherein the cell is a non-embryonic cell.  
   
   
       52 . A method of restoring or enhancing spermatogenesis, comprising providing a bone marrow derived male germline stem cell, or its progenitor cell, to the testes of a male subject, wherein the cell engrafts into the seminiferous epithelium and differentiates into a sperm cell, thereby restoring or enhancing spermatogenesis.  
   
   
       53 . A method of restoring fertility to a male subject having undergone chemotherapy or radiotherapy, or both and who desires restored fertility, comprising administering a therapeutically effective amount of bone marrow derived male germline stem cells, or their progenitor cells, to the subject, wherein the cells engraft into the seminiferous epithelium and differentiate into sperm cells, thereby restoring fertility.  
   
   
       54 . A method of reducing the amount of bone marrow derived germline stem cells, or their progenitor cells, in a subject comprising contacting bone marrow derived germline stem cells, or their progenitor cells, in the subject with an agent that reduces cell proliferation, thereby reducing the amount of bone marrow derived germline stem cells, or their progenitor cells, in the subject.  
   
   
       55 . The method of  claim 54 , wherein the agent is selected from the group consisting of a transforming growth factor-β, bone morphogenic protein antagonist, Protein Related to DAN and Cerberus and Gremlin.  
   
   
       56 . A method of reducing the amount of bone marrow derived germline stem cells, or their progenitor cells, in a subject comprising contacting bone marrow derived germline stem cells, or their progenitor cells, in the subject with an agent that inhibits cell survival, thereby reducing the amount of bone marrow derived germline stem cells, or their progenitor cells, in the subject.  
   
   
       57 . The method of  claim 56 , wherein the agent that inhibits survival is selected from the group consisting of a pro-apoptotic tumor necrosis factor super family member, antagonist of pro-survival Bcl-2 family member function and ceramide.  
   
   
       58 . The method of  claim 57 , wherein the pro-apoptotic tumor necrosis factor super family member is selected from the group consisting of tumor necrosis factor-α, Fas-ligand and TRAIL.  
   
   
       59 . The method of  claim 57 , wherein the pro-survival Bcl-2 family member is selected from the group consisting of Bcl-2, Bcl-XL, Bcl-W, Mcl-1 and A1.  
   
   
       60 . A kit for reducing the amount of bone marrow derived germline stem cells, or their progenitor cells, comprising an agent of  claim 57 , and instructions for using the agent to inhibit cell survival of bone marrow derived germline stem cells, or their progenitor cells, thereby reducing the amount of bone marrow derived germline stem cells, or their progenitor cells.  
   
   
       61 . A method of reducing the amount of bone marrow derived germline stem cells, or their progenitor cells, in a subject comprising contacting bone marrow derived germline stem cells, or their progenitor cells, in the subject with an agent that promotes cell death, thereby reducing the amount of bone marrow derived germline stem cells, or their progenitor cells, in the subject.  
   
   
       62 . The method of  claim 61 , wherein the agent that promotes cell death is selected from the group consisting of a pro-apoptotic tumor necrosis factor superfamily member, agonist of pro-apoptotic Bcl-2 family member function and ceramide.  
   
   
       63 . The method of  claim 62 , wherein the pro-apoptotic tumor necrosis factor superfamily member is selected from the group consisting of TNF alpha, Fas-ligand and TRAIL.  
   
   
       64 . The method of  claim 62 , wherein the pro-apoptotic Bcl-2 family member is selected from the group consisting of BAX, BAK, BID, HRK, BOD, BIM, NOXA, PUMA, BOK and BCL-XS.  
   
   
       65 . A kit for reducing the amount of bone marrow derived germline stem cells, or their progenitor cells, comprising an agent of  claim 62 , and instructions for using the agent to promote cell death of bone marrow derived germline stem cells, or their progenitors, thereby reducing the amount of bone marrow derived germline stem cells, or their progenitor cells.  
   
   
       66 . The method of claims  54 ,  56  or  61 , wherein the subject has a precancerous or cancerous condition.  
   
   
       67 . The method of  claim 66 , wherein the cancerous condition is a germ cell tumor, ovarian cancer or teratoma.  
   
   
       68 . A method of providing contraception to a subject comprising contacting bone marrow derived germline stem cells, or their progenitor cells, with an agent that decreases the amount of bone marrow derived germline stem cells, or their progenitor cells, thereby providing contraception to the subject.  
   
   
       69 . A kit for contraception in a subject comprising an agent of  claim 68 , and instructions for using the agent to decrease the amount of bone marrow derived germline stem cells, or their progenitor cells, thereby providing contraception to the subject.