Method for expansion of stem cells

Abstract

A method of increasing the growth of stem cells by mixing the stem cells with a growth medium that has been conditioned by an incubation with placental tissue. The method increases the expansion of the stem cell population.

1 . A method for the expansion or growth of stem cells without substantially inducing differentiation, comprising: incubating at least a portion of a placenta in a growth medium to condition said medium, and contacting at least one stem cell with said growth medium.  
   
   
       2 . The method of  claim 1 , wherein said at least one stem cell is totipotent, capable of differentiating into cells of all histological types of the body.  
   
   
       3 . The method of  claim 1 , wherein said at least one stem cell is pluripotent, capable of differentiating into numerous cells of the body, but not all.  
   
   
       4 . The method of  claim 1 , wherein said at least one stem cell is a progenitor cell, capable of differentiating into a restricted tissue type.  
   
   
       5 . The method of  claim 2 , wherein said totipotent stem cell is selected from the group consisting of: an embryonic stem cell, an extra-embryonic stem cell, a cloned stem cell, and a parthenogenesis derived cell.  
   
   
       6 . The method of  claim 3 , wherein said pluripotent stem cell is selected from the group consisting of a hematopoietic stem cell, an adipose stem cell, a mesenchymal stem cell, a cord blood stem cell, a placental stem cell, an exfoliated tooth derived stem cell, a hair follicle stem cell and a neural stem cell.  
   
   
       7 . The method of  claim 4 , wherein said progenitor stem cell is selected from the group consisting of neuronal, hepatic, nephrogenic, adipogenic, osteoblastic, osteoclastic, alveolar, cardiac, intestinal, and endothelial progenitor cells.  
   
   
       8 . The method of  claim 5 , wherein said embryonic stem cell expresses at least one marker selected from the group consisting of: stage-specific embryonic antigens (SSEA) 3, SSEA 4, Tra-1-60 and Tra-1-81, Oct-3/4, Cripto, gastrin-releasing peptide (GRP) receptor, podocalyxin-like protein (PODXL), and human telomerase reverse transcriptase (hTERT).  
   
   
       9 . The method of  claim 6 , wherein said hematopoietic stem cell expresses at least one marker selected from the group consisting of: CD34, c-kit, and the multidrug resistance transport protein (ABCG2).  
   
   
       10 . The method of  claim 6 , wherein said adipose-derived stem cell expresses at least one marker selected from the group consisting of: CD13, CD29, CD44, CD63, CD73, CD90, CD166, Aldehyde dehydrogenase (ALDH), and ABCG2.  
   
   
       11 . The method of  claim 6 , wherein said mesenchymal stem cell expresses at least one marker selected from the group consisting of: STRO-1, CD105, CD54, CD106, HLA-I markers, vimentin, ASMA, collagen-1, and fibronectin, but not HLA-DR, CD117, and hemopoietic cell markers.  
   
   
       12 . The method of  claim 6 , wherein said cord blood stem cell expresses at least one marker selected from the group consisting of: CD34, c-kit, and CXCR-4.  
   
   
       13 . The method of  claim 6 , wherein said placental stem cell expresses at least one marker selected from the group consisting of: Oct-4, Rex-1, CD9, CD13, CD29, CD44, CD166, CD90, CD105, SH-3, SH-4, TRA-1-60, TRA-1-81, SSEA-4 and Sox-2.  
   
   
       14 . The method of  claim 6 , wherein said exfoliated deciduous tooth stem cell expresses at least one marker s elected from the group consisting of: STRO-1, CD146 (MUC 18), alkaline phosphatase, MEPE, and bFGF.  
   
   
       15 . The method of  claim 7 , wherein said neural stem cell is characterized by expression of RC-2, 3CB2, BLB, Sox-2hh, GLAST, Pax 6, nesting, Muashi-1, and prominin.  
   
   
       16 . The method of  claim 1 , wherein said placenta is derived from a mammal.  
   
   
       17 . The method of  claim 1 , wherein said placenta is derived from a human.  
   
   
       18 . The method of  claim 1 , wherein said placenta is derived preterm.  
   
   
       19 . The method of  claim 1 , wherein said placenta is derived at term.  
   
   
       20 . The method of  claim 1 , wherein said placenta is perfused for a period of time with a cell culture media.  
   
   
       21 . The method of  claim 1 , wherein said cell culture media is supplemented with at least one growth factor.  
   
   
       22 . The method of  claim 21 , wherein said at least one growth factor is selected from the group consisting of: a WNT signaling agonist, TGF-b, bFGF, IL-6, SCF, BMP-2, thrombopoietin, EPO, IGF-1, IL-11, IL-5, Flt-3/Flk-2 ligand, fibronectin, LIF, HGF, NFG, angiopoietin-like 2 and 3, G-CSF, GM-CSF, Tpo, Shh, Wnt-3a, Kirre, and a mixture thereof.  
   
   
       23 . The method of  claim 1  wherein said media is capable of maintaining viability of a substantial portion of the placental tissue during the perfusion process.  
   
   
       24 . The method of  claim 1 , wherein said media is selected from the group consisting of Roswell Park Memorial Institute (RPMI-1640), Dublecco's Modified Essential Media (DMEM), Eagle's Modified Essential Media (EMEM), Optimem, and Iscove's Media.  
   
   
       25 . The method of  claim 1 , wherein a source of serum is added to the media.  
   
   
       26 . The method of  claim 1 , wherein the concentration of serum in the media is approximately between 0.1% to 25%.  
   
   
       27 . The method of  claim 1 , wherein the concentration of serum in the media is approximately 10%.  
   
   
       28 . The method of  claim 1 , wherein said serum is selected from the group consisting of: adult human serum, fetal human serum, fetal calf serum and umbilical cord blood serum.  
   
   
       29 . The method of  claim 1 , wherein said contacting step occurs after said incubating step.  
   
   
       30 . The method of  claim 1 , wherein said contacting step occurs simultaneously with said incubating step.  
   
   
       31 . The method of  claim 1 , wherein said incubating step occurs from about 1 second to about 3 weeks.  
   
   
       32 . The method of  claim 1 , wherein said incubating step occurs from about 24 hours to about 10 days.  
   
   
       33 . The method of  claim 1 , wherein said contacting step occurs from about 1 second to about 3 weeks.  
   
   
       34 . The method of  claim 1 , wherein said contacting step occurs from about 24 hours to about 10 days.  
   
   
       35 . The method of  claim 1 , wherein said placenta is a hemochorial, epitheliochorial, or endotheliochorial placenta.  
   
   
       36 . The method of  claim 1 , wherein said placenta is a hemochorial placenta.  
   
   
       37 . The method of  claim 1 , wherein perfusion is accomplished through the use of a perfusion apparatus cannulated to blood vessels connected to the placental body.  
   
   
       38 . The method of  claim 37 , wherein said perfusion apparatus allows for control of intravasular pressure, oxygen content, carbon dioxide content, pH, and flow rate of the perfused media flowing through said placental blood vessels.  
   
   
       39 . The method of  claim 38 , wherein the intravasular pressure of the perfusate is maintained at 30-80 Hg.  
   
   
       40 . The method of  claim 39 , wherein the intravasular pressure of the perfusate is maintained at 60 Hg.  
   
   
       41 . A stem cell with the preserved ability to proliferate but having a block in differentiation state induced by culturing in media conditioned by perfusion through a live placenta.  
   
   
       42 . The stem cell of  claim 41 , wherein said stem cell is selected from the group consisting of: a totipotent stem cell, a pluripotent stem cell, and a progenitor stem cell.  
   
   
       43 . The stem cell of  claim 41 , said stem cell is maintained in contact with the conditioned media for a period of 1 second to 3 weeks.  
   
   
       44 . The stem cell of  claim 41 , wherein said stem cell is maintained in contact with the conditioned media for a period of 1 second to 3 weeks.  
   
   
       45 . The stem cell of  claim 41 , wherein said stem cell is maintained in contact with the conditioned media for a period of 2 hours to 72 hours.  
   
   
       46 . The stem cell of  claim 41 , wherein said stem cell is maintained in contact with the conditioned media for a period of 12 hours to 24 hours.  
   
   
       47 . The stem cell of  claim 41 , wherein said stem cell is maintained in contact with the conditioned media in a living organism  
   
   
       48 . The stem cell of  claim 41 , wherein said contact between the conditioned media and the stem cell is prolonged by formulating the conditioned media in a slow release delivery system.  
   
   
       49 . The stem cell of  claim 41 , wherein said stem cells is initially cultured in contact with said placentally conditioned media for a period of time, subsequently to which it is cultured in a second culture with a different concentration of placentally conditioned media and an identical or variable mix of cytokines.  
   
   
       50 . The stem cell of  claim 49 , wherein said stem cell is initially cultured for 48 hours in a concentration of 20-100% placentally conditioned media, whereas in subsequent cultures it is maintained in a concentration of 0-50% conditioned media.  
   
   
       51 . The stem cell of  claim 50 , wherein said stem cell is maintained in a cell culture media that is supplemented with at least one growth factor selected from the group consisting of: WNT signaling agonist, TGF-b, bFGF, IL-6, SCF, BMP-2, thrombopoietin, EPO, IGF-1, IL-11, IL-5, Flt-3/Flk-2 ligand, fibronectin, LIF, HGF, NFG, angiopoietin-like 2 and 3, G-CSF, GM-CSF, Tpo, Shh, Wnt-3a, Kirre, and a mixture thereof.  
   
   
       52 . The stem cell of  claim 51 , wherein said stem cell is maintained in a 50% by volume placentally conditioned DMEM media with the following growth factors also in DMEM media: IL-3 (about 20 ng/ml), IL-6 (about 250 ng/ml), SCF (about 10 ng/ml), TPO (about 250 ng/ml), flt-3L (about 100 ng/ml).  
   
   
       53 . The stem cell of  claim 52 , wherein said stem cell is maintained in the presence of an agent selected from one or more of the following: an inhibitor of GSK-3, an inhibitor of histone deacetylase activity, and an inhibitor of DNA methyltransferase activity.  
   
   
       54 . The stem cells of  claim 52 , wherein said stem cell is rejuvenated by at least one procedure selected from the group consisting of: fusion with a more primitive stem cell, transfer of cytoplasm from a more primitive stem cells, and transfer of karyoplastic extracts from a more primitive stem cell.  
   
   
       55 . A method of treating degenerative diseases through administration of a composition of matter derived from media conditioned by a live placenta.  
   
   
       56 . The method of  claim 55 , wherein said degenerative disease effects a tissue selected from the group consisting of: smooth muscle tissue, striated muscle tissue, cardiac muscle tissue, bone tissue, bone spongy tissue, nervous system tissue, cartilage tissue, pancreatic ductal tissue, spleen tissue, thymus tissue, tonsil tissue, Peyer's patch tissue, lymph nodes tissue, thyroid tissue, epidermis tissue, dermis tissue, subcutaneous tissue, heart tissue, lung tissue, vascular tissue, endothelial tissue, blood cells, bladder tissue, kidney tissue, digestive tract tissue, esophagus tissue, stomach tissue, small intestine tissue, large intestine tissue, adipose tissue, uterus tissue, eye tissue, lung tissue, testicular tissue, ovarian tissue, prostate tissue, connective tissue, endocrine tissue, and mesentery tissue.  
   
   
       57 . The method of  claim 55 , wherein said placenta conditioned media is administered in combination with an agent capable of inducing stem cell expansion.  
   
   
       58 . The method of  claim 55 , wherein said placenta conditioned media is administered in combination with an agent capable of inducing stem cell differentiation into cells of the tissue in need of repair.  
   
   
       59 . The method of  claim 57 , wherein said agent capable of inducing stem cell expansion is selected from the group consisting of: TPO, SCF, IL-1, IL-3, IL-7, flt-3L, G-CSF, GM-CSF, Epo, FGF-1, FGF-2, FGF-4, FGF-20, VEGF, activin-A, IGF, EGF, NGF, LIF, PDGF, and a member of the bone morphogenic protein family.  
   
   
       60 . The method of  claim 58 , wherein said agent capable of inducing stem cell differentiation is selected from the group consisting of: HGF, cardiotrophin, BDNF, VEGF, FGF1, FGF2, FGF4, and FGF 20.  
   
   
       61 . The method of  claim 55 , wherein said placental conditioned media is concentrated to a sufficient extent to allow systemic administration while retaining biological effects.  
   
   
       62 . The method of  claim 61 , wherein said placental conditioned media is calibrated for specific Units of Activity based on a desired biological property.  
   
   
       63 . The method of  claim 62 , wherein said biological activity is the ability to stimulate proliferation of a defined culture of CD34 stem cells by 50%.  
   
   
       64 . The method of  claim 61 , wherein placentally conditioned media is administered according to biomarkers of stem cell activity in the patient in need of treatment.  
   
   
       65 . The method of  claim 62 , wherein said biomarker may be either an indicator of disease activity, or an indicator of stem cell regeneration.  
   
   
       66 . The method of  claim 61 , wherein a clinically applicable agent that possesses stem cell mobilizing activity is administered in conjunction with the placentally conditioned media and/or the stem cell proliferation inducing growth factor, and/or the inducer of stem cell differentation.  
   
   
       67 . The method of  claim 66 , wherein said stem cell mobilizing agent may be an antibody, a small molecule, or a protein.  
   
   
       68 . The method of  claim 67 , wherein said stem cell mobilizing agent is an antibody to CXCR-4.  
   
   
       69 . The method of  claim 67 , wherein said stem cell mobilizing agent is either a small molecular inhibitor of CXCR-4, or a statin.  
   
   
       70 . The method of  claim 67 , wherein said stem cell mobilizing agent is either a cytotoxic chemotherapy known to mobilize stem cells, or a growth factor such as G-CSF.  
   
   
       71 . The method of  claim 61 , wherein a dedifferentiation agent is used for expanding the differentiation potential of said stem cells.  
   
   
       72 . The method of  claim 71 , wherein said dedifferentiation agent is either an inhibitor of the enzyme GSK-3, and inhibitor of the histone deacetylase family of enzymes, or an inhibitor of DNA methyltransferase activity.  
   
   
       73 . The method of  claim 72 , wherein said dedifferentiation agents may be trichostatin A, valproic acid, buphenyl, or 5-azacytidine.  
   
   
       74 . A method of treating degenerative diseases, comprising administering a differentiating agent to selectively expand a population of pluripotent or progenitor cells, while concurrently administering live placental conditioned media in order to induce proliferation of the committed stem cell.  
   
   
       75 . A method of expanding stem cells that have been therapeutically reprogrammed, comprising contacting said cells with media that has been conditioned by a live placenta.  
   
   
       76 . The method of  claim 75  wherein said therapeutic reprogramming is accomplished by introduction into the target cell to be reprogrammed agents capable of acting at the epigenetic level to modify the cellular transcriptosome into a desired phenotype.  
   
   
       77 . The method of  claim 76 , whereby said target cell is fused with another cell of a more primitive state of differentiation.  
   
   
       78 . The method of  claim 75 , whereby said cell is temporarily permeabilized and cytoplasmic and/or karyoplasmic extracts are introduced into said cell from another cell of a more primitive state of differentiation.  
   
   
       79 . A method of accelerating hematopoietic recovery in a patient in need thereof, comprising administering placentally conditioned media.  
   
   
       80 . The method of  claim 79 , wherein said patient has been treated with chemotherapy, and/or radiotherapy with the scope of either ablating or diminishing the immune system.  
   
   
       81 . The method of  claim 79 , wherein said patient has been treated with chemotherapy, and/or radiotherapy with the scope of eradicating or ameliorating a malignancy.  
   
   
       82 . The method  claim 79 , wherein said patient has been induced into a state of reduced hematopoiesis as a result of chemical or radiation poisoning.  
   
   
       83 . The method of  claim 79 , wherein said patient is not administered a cellular graft to enhance recovery of the hematopoietic system.  
   
   
       84 . The method of  claim 79 , wherein said patient was administered either cord blood derived, peripheral blood derived, or bone marrow derived hematopoietic stem cells or progenitors thereof.  
   
   
       85 . The method of  claim 79 , wherein said patient is administered placentally conditioned media intravenously at a concentration sufficient to accelerate recovery of early hematopoietic progenitors.  
   
   
       86 . The method of  claim 79 , wherein said patient is administered placentally conditioned media at a concentration of 10-500 Units of placentally conditioned media per kilogram per day, said Units based on a logarithmic scale in which 1 Unit is sufficient to stimulated proliferation of a defined cell culture of CD34+ cells by 100% compared to control media.  
   
   
       87 . The cell culture of  claim 86 , wherein 1 Unit is defined on a logarithmic scale as the amount of placentally conditioned media needed to stimulate proliferation of a 200 μL culture of 5×10 3  human cord blood isolated CD34+.  
   
   
       88 . The method of  claim 79 , wherein said patient is treated intravenously, or through other means, with placental conditioned media for a period of time needed to obtain a granulocyte count of 500/mm 3 .  
   
   
       89 . The method of  claim 88 , wherein said patient is treated intravenously, or through other means, with placental conditioned media for a period of time between 7 days to 15 days.  
   
   
       90 . The method of  claim 79 , wherein a growth factor is concurrently given with the administration of placentally conditioned media.  
   
   
       91 . The method of  claim 90 , wherein said growth factor is selected from the group consisting of G-CSF, pegylated G-CSF, TPO, IL-11, GM-CSF, and flt-3L.  
   
   
       92 . A method of treating patient with tissue ischemia through induction of endothelial stem cell expansion using placentally conditioned media.  
   
   
       93 . The method of  claim 92 , wherein said ischemia is present in at least one tissue selected from the group consisting of: smooth muscle tissue, striated muscle tissue, cardiac muscle tissue, bone tissue, bone spongy tissue, nervous system tissue, cartilage tissue, pancreatic ductal tissue, spleen tissue, thymus tissue, tonsil tissue, Peyer's patch tissue, lymph nodes tissue, thyroid tissue, epidermis tissue, dermis tissue, subcutaneous tissue, heart tissue, lung tissue, vascular tissue, endothelial tissue, blood cells, bladder tissue, kidney tissue, digestive tract tissue, esophagus tissue, stomach tissue, small intestine tissue, large intestine tissue, adipose tissue, uterus tissue, eye tissue, lung tissue, testicular tissue, ovarian tissue, prostate tissue, connective tissue, endocrine tissue, and mesentery tissue.  
   
   
       94 . The method of  claim 93 , wherein said ischemia is presenting as advanced angina.  
   
   
       95 . The method of  claim 94 , wherein placentally conditioned media is concentrated and administered into the ischemic myocardium using the minithoracotomy procedure.  
   
   
       96 . The method of  claim 94 , wherein placentally conditioned media is concentrated and administered into the ischemic myocardium using the NOGA electromagnetic mapping and injection system.  
   
   
       97 . The method of  claim 94 , wherein placentally conditioned media is concentrated and administered into the ischemic myocardial area using a balloon catheter.  
   
   
       98 . The method of  claim 94 , wherein a secondary agent is added that is capable of inducing proliferation of differentiated and undifferentiated endothelial cells.  
   
   
       99 . The method of  claim 98 , wherein said secondary agent is either a nucleic acid, a protein, or a small molecule.  
   
   
       100 . The method of  claim 99 , wherein said secondary agent is plasmid DNA encoding a polypeptide selected from the group consisting of: HIF-1, VEGF, FGF1, FGF2, FGF4, FGF20, and angiopoietin.  
   
   
       101 . The method of  claim 99 , wherein said secondary agent is selected from the group consisting of: VEGF, FGF1, FGF2, FGF4, FGF20, and angiopoietin.  
   
   
       102 . The method of  claim 94 , wherein an exogenous source of stem cells are delivered into the ischemic area.  
   
   
       103 . The method of  claim 94 , wherein an endogenous source of stem cells are delivered into the ischemic area.  
   
   
       104 . The method of  claim 102 , wherein exogenous stem cells may be autologous or allogenenic mesenchymal, adipose, endothelial, bone marrow, mobilized peripheral blood, umbilical, or artificially reprogrammed stem cells.  
   
   
       105 . The method of  claim 103 , wherein endogenous stem cells are mobilized with a mobilization agent.  
   
   
       106 . The method of  claim 92 , wherein said patient suffering from ischemia is a victim of Critical Limb Ischemia.  
   
   
       107 . The method of  claim 92 , wherein said patient is administered a combination of placentally conditioned media intramuscularly in the area of ischemia as detected by angiography.  
   
   
       108 . The method of  claim 107 , wherein autologous or allogenenic mesenchymal, adipose, endothelial, bone marrow, mobilized peripheral blood, umbilical, or artificially reprogrammed stem cells are injected with the placentally conditioned media in a localized environment intramuscularly.  
   
   
       109 . The method of  claim 108 , wherein autologous lymphocytes are injected with said stem cell source in order to synergize with the placentally conditioned media and the injected stem cells.  
   
   
       110 . The method of  claim 92 , wherein said patient has suffered from a cerebral ischemia.  
   
   
       111 . The method of  claim 110 , wherein said patient is treated immediately after the ischemia episode or in a period of time subsequently.  
   
   
       112 . A method of culturing a placenta in its original 3-dimensional structure in such a manner to reproduce the in vivo environment in which it resides in the pregnant women thus retaining capability of generation and secretion of growth factors and proteins that maintain the fetal regenerative capacity, said method comprising the steps of: 
 acquiring a placenta under sterile conditions;    cannulating blood vessels of the placenta in order to allow proper perfusion in circumstances similar to as if the placenta was performing its in vivo functions;    perfusing said placenta with a nutrient mix, in a buffer that would mimic physiological conditions;    maintaining a temperature and physical environment similar to that found in the pregnant woman's body; and    imitating conditions of flow, pH, oxygenation, and pressure similar to that found in the body.    
   
   
       113 . The method of  claim 112 , wherein perfusion of both the maternal and fetal circulatory components of the placenta is performed.  
   
   
       114 . The method of  claim 112 , wherein a nutrient mixture is used that possesses similar nutrient requirements as the fetal and maternal circulation, respectively.  
   
   
       115 . The method of  claim 112 , wherein a temperature of 37° C. is maintained during the perfusion process.  
   
   
       116 . The method of  claim 112 , wherein pH is monitored by the perfusion apparatus in a real-time basis, and adjusted using adequate quantities of acids, bases, or buffers.  
   
   
       117 . The method of  claim 112 , wherein oxygen content is maintained similar to that found in the fetal and maternal circulatory contribution to the placenta.  
   
   
       118 . The method of  claim 117 , wherein oxygen content may be increased through the use of adding natural or artificial oxygen carriers to the perfusion solution.  
   
   
       119 . The method of  claim 112 , wherein an oxygenator may be attached to the perfusion apparatus, in conjunction with, or separately, from an oxygen sensor, said combination being used to adjust in real-time oxygen content.  
   
   
       120 . The method of  claim 112 , wherein osmolality is maintained through the use of known means such as addition of albumin or colloids to the perfusion solution.  
   
   
       121 . A method of producing a cosmetic for topical use in rejuvenating aged skin comprising the steps of: 
 concentrating placentally conditioned media;    quantifying and standardizing biological effect of said media, and    formulating said media in a carrier solution that is suitable for transdermal delivery.    
   
   
       122 . The method of  claim 121 , wherein said media is selected from the group consisting of: a physiological buffer, a media capable of maintaining cellular viability, and a media enriched in nutrients and mimicking the content of the maternal/fetal circulation.  
   
   
       123 . The method of  claim 122 , wherein said media is selected from the group consisting of DMEM, RPMI, and saline USP.  
   
   
       124 . The method of  claim 123 , wherein said media is DMEM.  
   
   
       125 . The method of  claim 122 , wherein said media contains an anticoagulant at sufficient quantities to inhibit clotting during placental perfusion.  
   
   
       126 . The method of  claim 121 , wherein said quantification is based on ability of placentally conditioned media to induce proliferation of dermal stem cells.  
   
   
       127 . The method of  claim 121 , wherein a moisturizing agent is added to the cosmetic preparation.  
   
   
       128 . The method of  claim 121 , wherein said carrier further comprises at least one nutrient to replenish the skin.  
   
   
       129 . The method of  claim 121 , wherein said carrier contains at least one anti-oxidant compound.