WO2014123930A2 - Prévention et traitement de fibrose de tissu - Google Patents

Prévention et traitement de fibrose de tissu Download PDF

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WO2014123930A2
WO2014123930A2 PCT/US2014/014733 US2014014733W WO2014123930A2 WO 2014123930 A2 WO2014123930 A2 WO 2014123930A2 US 2014014733 W US2014014733 W US 2014014733W WO 2014123930 A2 WO2014123930 A2 WO 2014123930A2
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cells
tissue
stem cells
cell
group
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PCT/US2014/014733
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WO2014123930A3 (fr
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Joseph L. Williams
Paul A. Lucas
Brian S. Murphy
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Williams Joseph L
Lucas Paul A
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/48—Reproductive organs
    • A61K35/54—Ovaries; Ova; Ovules; Embryos; Foetal cells; Germ cells
    • A61K35/545—Embryonic stem cells; Pluripotent stem cells; Induced pluripotent stem cells; Uncharacterised stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0667—Adipose-derived stem cells [ADSC]; Adipose stromal stem cells

Definitions

  • the invention pertains to the field of inhibiting fibrosis, more particularly, to inhibition of surgical adhesions. More particularly, the invention provides means of utilizing combinations of cells, products derived from cells, and combination of various compositions of matter for enhancing therapeutic efficacy in prevention and/or treatment of surgical adhesions.
  • fibrous tissue is part of the normal healing process after injury, including injury due to surgery. It is often referred to as "scar” or “scar tissue".
  • the primary component of fibrous tissue is fibroblasts.
  • scar tissue does not have any identical properties of the tissue it replaces, thus it often interferes with the normal function of the affected tissue.
  • One particular case is surgical adhesions.
  • the normal mesothelial cells lining the peritoneum and surface of the abdominal organs is replaced by scar tissue.
  • This scar tissue can form connections between the peritoneum and abdominal organs or between the abdominal organs - adhesions. These adhesions can disrupt the normal function of the abdominal organs, i.e. strangulation of the small intestines.
  • the formation of scar tissue is a reactive process, and several different factors can apparently modulate the pathways leading to tissue fibrosis. Such factors include the early inflammatory responses, local increase in fibroblast cell populations, modulation of the synthetic function of fibroblasts, and altered regulation of the biosynthesis and degradation of collagen. Stimulation of fibroblast activity is involved in the development of fibrotic conditions, including spontaneous and induced conditions. Abnormal accumulation of collagen in the extracellular matrix, resulting from excessive fibroblast proliferation and/or collagen production, can cause fibrosis of a number of tissues including the skin. Many common debilitating diseases, such as liver cirrhosis and pulmonary fibrosis, involve the proliferation of fibrous tissue as do certain skin diseases such as scleroderma, and the formation of surgical adhesions.
  • Peritoneal adhesions are fibrotic bands that form between surfaces in the peritoneal cavity. The most important stimulant of the formation of adhesions is trauma on the peritoneum. Adhesions usually arise due to intra-abdominal surgery. In almost all of the intra-abdominal interventions, adhesions occur with varying severity. Surgically- associated peritoneal adhesions are reported as the cause of approximately 32% of acute intestinal obstruction and 65-75% of all small bowel obstructions. It is believed that peritoneal adhesions develop after 93-100% of upper abdominal laparotomies and after 67-93% of lower abdominal laparotomies. As many as 15-18% of surgical procedure- associated adhesions require surgical re-intervention.
  • Peritoneal adhesions appearing after abdominal and pelvic operations may lead to further pathological states, such as chronic abdominal pain and infertility [5].
  • the visceral peritoneum is composed of a single layer of mesothelial cells placed on the basement membrane.
  • Basement membranes are the layer in which mesothelial cells are attached to the tissue below with loose connective tissue. Due to its thin and delicate structure, the peritoneum is quite susceptible to trauma [6].
  • mesothelial cells are harmed and shed. In the region where these cells are shed, initiation of the inflammatory cascade occurs and serous exudate is formed. In some cases, as a consequence of the organization of fibrinogen found in exudate, fibrin bands are formed between the damaged mesothelial layers [7].
  • fibrin bands are formed between the damaged mesothelial layers [8].
  • the invention provides cellular populations, combinations of cells with various compositions of matter, and products derived from cells for prevention of surgical adhesions.
  • the invention teaches the use of mesenchymal stem cell populations as an intervention to inhibit formation of adhesions.
  • the combination of pluripotent adult stem cells with Seprafilm, other possible matrices, or other antifibrotic agents is provided.
  • Specific embodiments include the combination of various cell populations together with growth factors, such as provided by platelet rich plasma.
  • conditioned media from mesenchymal stem cells is utilized to inhibit formation of surgical adhesions.
  • Various combinations are described that include means of conditioning cells prior to implantation.
  • implantation of the therapeutic composition is performed by injecting a suspension of stem cells into the peritoneal cavity just prior to, or immediately after, closure of the surgical incision.
  • the stem cells free floating in the peritoneal cavity, will adhere to any site where the mesothelium has been abraded or has necrosed.
  • stem cells are placed at, near, or in the fibrous tissue (including normal or abnormal fibrous tissue) or tissue at risk for progressing into fibrous tissue, resulting in minimal physical and psychological trauma to the patient.
  • the therapeutic composition can be administered in the same cannula or needle without the need to reposition it several times.
  • the therapeutic composition can directly or indirectly induce the degradation, shrinkage, relaxation, or stretching of the fibrous tissue or tissue at risk for progression into fibrous tissue.
  • stem cells are administered with the intent of differentiating into mesothelial cells in vivo, and thus preventing attachment of fibroblasts to peritoneum or internal organs
  • stem cells are differentiated in vitro to mesothelial cells prior to administration.
  • devices and methods are provided that allow the therapeutic composition to be retrieved if implanted at the wrong site because the therapeutic composition will not be caused to release the antifibrotic agent until the device is implanted in the desired location.
  • the therapeutic composition is in conjuction with a biodegradable matrix.
  • pluripotent cells of muscle or bone marrow such as cells described in US patent #7,259,011 and incorporated here by reference, are used.
  • a therapeutic composition for inhibiting development of fibrosis in a patient in need of such treatment, the therapeutic composition being biodegradable and implantable at, near or in the area of risk for development of fibrotic tissue, the therapeutic composition comprising an antifibrotic agent configured to immediately release an effective amount of the anti-fibrotic agent within 24 hours, said anti-fibrotic agent comprising of a therapeutic cell population.
  • said therapeutic compositions are comprised of either differentiated mesothelial cells or any stem cell capable of differentiating in vivo into mesothelial cells.
  • Such stem cells include: adherent cells derived from bone marrow, muscle, skin, or other tissues, said cells possessing a marker selected from a group of markers consisting of: CD90, CD 105, CD9, CD73, CD27, and CD44; cells are of the mesenchymal lineage; . isolated human pluripotent adult stem cells which express CD13, CD34, CD56 and CD117, and do not express CD10; . isolated human pluripotent adult stem cells which express CD13, CD34, CD56 and CD117, and do not express CD10 and further do not express CD2, CD5, CD14, CD19, CD33, CD45, and DRII; isolated endometrial regenerative cells, etc..
  • the therapeutic composition is comprised of any of the listed stem cells which have been induced to differentiate, or initiate differentiation to, mesothelial cells.
  • stem cells which have been induced to differentiate, or initiate differentiation to, mesothelial cells.
  • factors must be taken into consideration, such as: ability for ex vivo expansion without loss of therapeutic activity, ease of extraction, general potency of activity, and potential for adverse effects.
  • Ex vivo expansion ability of stem cells can be measured using typical proliferation and colony assays known to one skilled in the art, while identification of therapeutic activity depends on functional assays that test biological activities such as: ability to support mesothelial function and ability inhibit fibrosis.
  • Assessment of therapeutic activity can also be performed using surrogate assays which detect markers associated with a specific therapeutic activity.
  • markers include CD34 or CD 133, which are associated with stem cell activity and ability to support angiogenesis [9].
  • Other assays useful for identifying therapeutic activity of stem cell populations for use with the current invention include evaluation of production of factors associated with the therapeutic activity desired. For example, identification and quantification of production of FGF, VEGF, angiopoietin, or other such angiogenic molecules may be used to serve as a guide for approximating therapeutic activity in vivo [10]. Additionally, secretion of factors that inhibit smooth muscle atrophy or neuronal dysfunction may also be used as a marker for identification of cells that are useful for practicing the current invention.
  • embryonic stem cells possess certain desirable properties, such as the ability to differentiate to almost every cell comprising the host. Additionally, embryonic stem cells secrete numerous factors capable of inhibiting the process of adhesion formation. Unfortunately, certain drawbacks exist that limit the utility of this cell type for widespread therapeutic implementation. The potential for carcinogenicity is apparent in that human embryonic stem cells administered to immunocompromised mice leads to formation of teratomas [11]. Accordingly, for use in the current invention, embryonic stem cells have to be either differentiated into a mesothelial cell, a stem cell, or a progenitor cell that is not capable of forming tumors.
  • embryonic stem cells useful the practice of the current invention should not differentiate in a substantial amount in an uncontrolled manner or into tissue which is pathological to the patient's well being. Although several technologies are currently being tested for selecting embryonic stem cells that do not cause teratomas, these methods are still in their infancy [12]. Therefore, one method of utilizing embryonic stem cells for the practice of this invention is to encapsulate said embryonic stem cells, or place said cells into a permeable barrier so as to allow for secretion of therapeutic factors elaborated by said cells without the risk of causing cancer or undesired tissue growth.
  • encapsulation technology has been successful in "semi-isolating" cells with therapeutic potential from the body, for examples of this the practitioner of the invention is referred to work on microencapsulation of islets for treatment of diabetes, in which cases xenogeneic islets are used [13, 14], or other systems of therapeutic cellular xenograft therapy [15-17].
  • Said encapsulated cells may be administered systemically, or in a preferred embodiment locally in an area proximal to the area of surgical injury.
  • encapsulated cells may be placed in a removable chamber in subcutaneous tissue similarly to the one described in US Pat No. 5,958,404.
  • the advantages of using a removable chamber is that administration of cell therapy is not a permanent intervention and may be withdrawn upon achievement of desired therapeutic effect, or at onset of adverse effects.
  • Another embodiment of the current invention is the use of embryonic stem cell supernatant as a therapy for prevention of surgical adhesions.
  • Specific embodiments include identification of substantially purified fractions of said supernatant capable of inducing mesothelial cell proliferation, and inhibition of fibrosis. Identification of such therapeutically active fractions may be performed using methods commonly known to one skilled in the art, and includes separation by molecular weight, charge, affinity towards substrates and other physico-chemical properties.
  • supernatant of embryonic stem cell cultures is harvested substantially free from cellular contamination by use of centrifugation or filtration.
  • Supernatant may be concentrated using means known in the art such as solid phase extraction using C18 cartridges (Mini-Spe-ed C18-14 , S.P.E. Limited, Concord ON). Said cartridges are prepared by washing with methanol followed by deionized-distilled water. Up to 100 ml of embryonic stem cell supernatant may be passed through each cartridge before elution. After washing the cartridges material adsorbed is eluted with 3 ml methanol, evaporated under a stream of nitrogen, redissolved in a small volume of methanol, and stored at 4.degree. C. Before testing the eluate for activity in vitro, the methanol is evaporated under nitrogen and replaced by culture medium.
  • Said C18 cartridges are used to adsorb small hydrophobic molecules from the embryonic stem cell culture supernatant, and allows for the elimination of salts and other polar contaminants. It may, however be desired to use other adsorption means in order to purify certain compounds from the embryonic stem cell supernatant. Said concentrated supernatant may be assessed directly for biological activities useful for the practice of this invention, or may be further purified. Further purification may be performed using, for example, gel filtration using a Bio-Gel P-2 column with a nominal exclusion limit of 1800 Da (Bio- Rad, Richmond Calif.).
  • Said column may be washed and pre-swelled in 20 mM Tris-HCl buffer, pH 7.2 (Sigma) and degassed by gentle swirling under vacuum.
  • Bio-Gel P-2 material be packed into a 1.5. times.54 cm glass column and equilibrated with 3 column volumes of the same buffer.
  • Embryonic stem cell supernatant concentrates extracted by C18 cartridge may be dissolved in 0.5 ml of 20 mM Tris buffer, pH 7.2 and run through the column. Fractions may be collected from the column and analyzed for biological activity.
  • Other purification, fractionation, and identification means are known to one skilled in the art and include anionic exchange chromatography, gas chromatography, high performance liquid chromatography, nuclear magnetic resonance, and mass spectrometry. Administration of supernatant active fractions may be performed locally or systemically.
  • embryonic stem cells are differentiated into endothelial progenitor cells in vitro, followed by administration to a patient in need of therapy at a concentration and frequency sufficient to ameliorate or cure surgical adhesions. Differentiation into endothelial progenitors may be performed by several means known in the art [18].
  • One such means includes generation of embryoid bodies through growing human embryonic stem cells in a suspension culture. Said embryoid bodies are subsequently dissociated and cells expressing endothelial progenitor markers are purified [19]. Purification of endothelial cells from embryoid bodies can be performed using, of example, selection for PECAM-1 expressing cells. Purified cells can be expanded in culture and used for injection. Another alternative method of generating endothelial progenitors from embryonic stem cells involves removing media from embryonic stem cells a period of time after said embryonic stem cells are plated and replacing said media with a media containing endothelial-differentiating factors.
  • EBM2 endothelial cell basal medium-2
  • endothelial cells, or endothelial precursor cells, generated from embryonic stem cells may be administered to the patient in an injection solution, which may be saline, mixtures of autologous plasma together with saline, or various concentrations of albumin with saline. Ideally pH of the injection solution is from about 6.4 to about 8.3, optimally 7.4.
  • Excipients may be used to bring the solution to isotonicity such as, 4.5% mannitol or 0.9% sodium chloride, pH buffers with art-known buffer solutions, such as sodium phosphate.
  • Other pharmaceutically acceptable agents can also be used to bring the solution to isotonicity, including, but not limited to, dextrose, boric acid, sodium tartrate, propylene glycol, polyols (such as mannitol and sorbitol) or other inorganic or organic solutes.
  • Injection can be performed systemically, with the goal of injected cells homing to penile tissues associated with ED, or alternatively administration may be local, via intraperitoneal administration.
  • an endothelial progenitor/endothelial cell chemoattractant factor may be used in order to increase the number of cells homing to the area of need.
  • Said chemoattractant factors may include SDF-1 and/or VEGF, various isoforms thereof and small molecule agonists of the VEGFR-1 and/or VEGFR2, and/or CXCR4.
  • Localization of said chemotactic factors to the area causative of ED may be performed using agents such as fibrin glue or certain delivery polymers known to one who is skilled in the art, these may include: polyvinyl chloride, polylactic acid (PLA), poly-L-lactic acid (PLLA), poly-D-lactic acid (PDLA), polyglycolide, polyglycolic acid (PGA), polylactide-co-glycolide (PLGA), polydioxanone, poly gluconate, polylactic acid-polyethylene oxide copolymers, polyethylene oxide, modified cellulose, collagen, polyhydroxybutyrate, polyhydroxpriopionic acid, polyphosphoester, poly(alpha-hydroxy acid), polycaprolactone, polycarbonates, polyamides, poly anhydrides, polyamino acids, polyorthoesters, polyacetals, polycyanoacrylates, degradable urethanes, aliphatic polyester polyacrylates, polymethacrylate, acyl substituted cellulose acetate
  • Acceptable carriers, excipients, or stabilizers are also contemplated within the current invention, said carriers, excipients and stabilizers being relatively nontoxic to recipients at the dosages and concentrations employed, and may include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid, n-acetylcysteine, alpha tocopherol, and methionine; preservatives such as hexamethonium chloride; octadecyldimethylbenzyl ammonium chloride; benzalkonium chloride; phenol, benzyl alcohol, or butyl; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexinol; 3-pentanol; and me-cresol); low molecular weight polypeptides; proteins, such as gelatin, or non-specific immunoglobulins; amino acids such as glycine, glutamine, asparag
  • embryonic stem cells are differentiated into a desired phenotype microencapsulated so as to retain viability and ability to produce growth factors, while at the same time escaping immune mediated killing.
  • This may be accomplished using known microencapsulation methods described in the art, such as described in US Patent # 7,041,634 to Weber et al, or US Patent Application # 20040136971 to Scharp et al.
  • embryonic stem cells may be irradiated either prior to, or subsequent to, encapsulation so as to block ability to proliferate while retaining growth factor producing activity.
  • embryonic stem cells are grown on the outside of a hollow-fiber filter which is connected to a continuous extracorporeal system.
  • Said hollow-fiber system contains pores in the hollow fiber of sufficient size so has to allow exchange of proteins between circulating blood cells and cultured cells on the outside of the hollow fibers, without interchange of host cells with the embryonic stem cells.
  • cord blood stem cells are administered systemically into a patient at risk of surgical adhesions.
  • Said cord blood stem cells may be administered as a heterogenous population of cells by the administration of cord blood mononuclear cells.
  • Said cells may be isolated according to many methods known in the art. In one particular method, cord blood is collected from fresh placenta and mononuclear cells are purified by centrifugation using a density gradient such as Ficoll or Percoll, in another method cord blood mononuclear cells are isolated from contaminating erythrocytes and granulocytes by the Hetastarch with a 6% (wt/vol) hydroxyethyl starch gradient.
  • cord blood stem cells are fractionated and the fraction with enhanced therapeutic activity is administered to the patient. Enrichment of cells with therapeutic activity may be performed using physical differences, electrical potential differences, differences in uptake or excretion of certain compounds, as well as differences in expression marker proteins. Distinct physical property differences between stem cells with high proliferative potential and low proliferative potential are known. Accordingly, in some embodiments of the invention, it will be useful to select cord blood stem cells with a higher proliferative ability, whereas in other situations, a lower proliferative ability may be desired.
  • cells are directly injected into the area of need, such as in the peritoneal cavity, in which case it will be desirable for said stem cells to be substantially differentiated, whereas in other embodiments, cells will be administered systemically and it this case with may be desirable for the administered cells to be less differentiated, so has to still possess homing activity to the area of need.
  • FACS Fluorescent Activated Cell Sorter
  • Other methods of separating cells based on physical properties include the use of filters with specific size ranges, as well as density gradients and pheresis techniques.
  • techniques such as electrophotoluminescence may be used in combination with a cell sorting means such as FACS.
  • Selection of cells based on ability to uptake certain compounds can be performed using, for example, the ALDESORT system, which provides a fluorescent-based means of purifying cells with high aldehyde dehydrogenase activity. Cells with high levels of this enzyme are known to possess higher proliferative and self-renewal activities in comparison to cells possessing lower levels.
  • cord blood cells are purified for certain therapeutic properties based on expression of markers.
  • cord blood cells are purified for the phenotype of endothelial precursor cells. Said precursors, or progenitor cells express markers such as CD133, and/or CD34.
  • Said progenitors may be purified by positive or negative selection using techniques such as magnetic activated cell sorting (MACS), affinity columns, FACS, panning, or by other means known in the art.
  • Cord blood derived endothelial progenitor cells may be administered directly into the target tissue for prevention of adhesions, or may be administered systemically. Another variation of this embodiment is the use of differentiation of said endothelial precursor cells in vitro, followed by infusion into a patient.
  • Verification for endothelial differentiation may be performed by assessing ability of cells to bind FITC-labeled Ulex europaeus agglutinin- 1 , ability to endocytose acetylated Di-LDL, and the expression of endothelial cell markers such as PECAM-1, VEGFR-2, or CD31. Certain desired activities can be endowed onto said cord blood stem cells prior to administration into the patient.
  • cord blood cells may be "activated" ex vivo by a brief culture in hypoxic conditions in order to upregulate nuclear translocation of the HIF-1 transcription factor and endow said cord blood cells with enhanced angiogenic potential.
  • Hypoxia may be achieved by culture of cells in conditions of 0.1% oxygen to 10% oxygen, preferably between 0.5% oxygen and 5% oxygen, and more preferably around 1% oxygen.
  • Cells may be cultured for a variety of timepoints ranging from 1 hour to 72 hours, more preferably from 13 hours to 59 hours and more preferably around 48 hours.
  • Assessment of angiogenic, and other desired activities useful for the practice of the current invention can be performed prior to administration of said cord blood cells into the patient. Assessment methods are known in the art and include measurement of angiogenic factors, ability to support viability and activity of cells associated with fibrosis, as well as ability to induce regeneration of said cellular components associated with surgical adhesions.
  • cord blood cells are cultured with an inhibitor of the enzyme GSK-3 in order to enhance expansion of cells with pluripotent characteristics while not increasing the rate of differentiation.
  • cord blood cells are cultured in the presence of a DNA methyltransferase inhibitor such as 5-azacytidine in order to endow a "de-differentiation" effect.
  • cord blood cells are cultured in the presence of a differentiation agent that skews said cord blood stem cells to generate enhance numbers of cells which are useful for prevention of surgical adhesions after said cord blood cells are administered into a patient.
  • placental stem cells may be purified directly from placental tissues, said tissues including the chorion, amnion, and villous stroma [21, 22].
  • placental tissue is mechanically degraded in a sterile manner and treated with enzymes to allow dissociation of the cells from the extracellular matrix.
  • enzymes include, but not restricted to trypsin, chymotrypsin, collagenases, elastase and/or hylauronidase. Suspension of placental cells are subsequently washed, assessed for viability, and may either be used directly for the practice of the invention by administration either locally or systemically.
  • cells may be purified for certain populations with increased biological activity. Purification may be performed using means known in the art, and described above for purification of cord blood stem cells, or may be achieved by positive selection for the following markers: SSEA3, SSEA4, TRA1-60, TRA1-81, c-kit, and Thy-1. In some situations it will be desirable to expand cells before introduction into the human body. Expansion can be performed by culture ex vivo with specific growth factors [23, 24]. The various embodiments of the invention described above for cord blood and embryonic stem cells can also be applied for placental stem cells. [0021] Deciduous teeth (baby teeth) have been recently identified as a source of pluripotent stem cells with ability to differentiate into endothelial, neural, and bone structures.
  • Said pluripotent stem cells have been termed "stem cells from human exfoliated deciduous teeth” (SHED).
  • SHED cells are administered systemically or locally into a patient with ED at a concentration and frequency sufficient for induction of therapeutic effect.
  • SHED cells can be purified and used directly, certain sub-populations may be concentrated, or cells may be expanded ex vivo under distinct culture conditions in order to generate phenotypes desired for maximum therapeutic effect. Growth and expansion of SHED has been previously described by others.
  • exfoliated human deciduous teeth are collected from 7- to 8-year-old children, with the pulp extracted and digested with a digestive enzyme such as collagenase type I.
  • Concentrations necessary for digestion are known and may be, for example 1 -5 mg/ml, or preferable around 3 mg/ml.
  • dispase may also be used alone or in combination, concentrations of dispase may be 1-10 mg/ml, preferably around 4 mg/ml.
  • Said digestion is allowed to occur for approximately 1 h at 37°C.
  • Cells are subsequently washed and may be used directly, purified, or expanded in tissue culture.
  • the various embodiments of the invention described above for cord blood and embryonic stem cells can also be applied for exfoliated teeth stem cells.
  • Amniotic fluid is routinely collected during amniocentesis procedures.
  • One method of practicing the current invention is utilizing amniotic fluid derived stem cells for treatment of surgical adhesions.
  • amniotic fluid mononuclear cells are utilized therapeutically in an unpurified manner.
  • Said amniotic fluid stem cells are administered either locally or systemically in a patient at risk of adhesion development.
  • amniotic fluid stem cells are substantially purified based on expression of markers such as SSEA-3, SSEA4, Tra-1-60, Tra-1-81 and Tra-2- 54, and subsequently administered.
  • cells are cultured, as described in US patent application # 20050054093, expanded, and subsequently infused into the patient.
  • Amniotic stem cells are described in the following references [25-27].
  • One particular aspect of amniotic stem cells that makes them amenable for use in practicing certain aspects of the current invention is their bi-phenotypic profile as being both mesenchymal and neural progenitors [28]. This property is useful for treatment of patients with ED in which neural regeneration is required to a greater extent than vascular repair.
  • the various embodiments of the invention described above for cord blood and embryonic stem cells can also be applied for amniotic fluid stem cells.
  • a wide variety of stem cells are known to circulate in the periphery. These include multipotent, pluripotent, and committed stem cells.
  • mobilization of stem cells is induced in order to increase the number of circulating stem cells, so that harvesting efficiency is increased. Said mobilization allows for harvest of cells with desired properties for practice of the invention without the need to perform bone marrow puncture.
  • methods to induce mobilization are known. Methods such as administration of cytotoxic chemotherapy, for example, cyclophosphamide or 5-fluoruracil are effective but not preferred in the context of the current invention due to relatively unacceptable adverse events profile.
  • Suitable agents useful for mobilization include: granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin 1 (IL-1), interleukin 3 (IL-3), stem cell factor (SCF, also known as steel factor or kit ligand), vascular endothelial growth factor (VEGF), Flt-3 ligand, platelet-derived growth factor (PDGF), epidermal growth factor (EGF), fibroblast growth factor-1 (FGF-1), fibroblast growth factor-2 (FGF-2), thrombopoietin (TPO), interleukin- 11 (IL-11), insulin-like growth factor-1 (IGF-1), megakaryocyte growth and development factor (MGDF), nerve growth factor (NGF), hyperbaric oxygen, and 3 -hydroxy-3 -methyl glutaryl coenzyme A (HMG CoA)reductase inhibitors.
  • G-CSF granulocyte colony stimulating factor
  • GM-CSF granulocyte
  • endometrial regenerative cells are used for treatment of surgical adhesions, generation of ERC is performed using menstrual blood as a starting source.
  • menstrual blood approximately 7 ml of menstrual blood is collected from female subjects after informed consent the second day after menstrual blood flow initiated. Collection is performed in a sterile urine cup or a Diva cup and then transferred into a 50 ml conical tube with 0.2 ml amphotericin B 0.2 ml penicillin/streptomycin (50 ug/ml) and 0.1 ml EDTA-Na2 in a total volume of 40 ml phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • the buffy coat is collected and placed into another 50 ml conical tube together with 40 ml of PBS.
  • Cells are centrifuged at 400 g for 10 minutes, after which the supernatant is decanted and the cell pellet is resuspended in 40 ml of PBS and centrifuged again for 10 minutes at 400 g.
  • the cell pellet is subsequently resuspended in 5 ml DMEM medium supplemented with 1% penicillin/streptomycin, 1% amphotericin B, 1% glutamine and 20% FBS (hereafter referred to as completed DMEM).
  • the resuspended cells are mononuclear cells substantially free of erythrocytes and polymorphonuclear leukocytes as assessed by visual morphology microscopically. Viability of the cells is assessed with trypan blue. For expansion, 1 million menstrual blood derived mononuclear cells are placed in a 15 ml sterile Petri dish (Corning, Acton, Mass.) in 10 ml complete DMEM medium. DMEM is a variation of MEM, and contains approximately four times as much of the vitamins and amino acids present in MEM and two to four times as much glucose as MEM.
  • tissue culture media may be used such as Roswell Park Memorial Institute Media (RPMI- 1640) which is available from Sigma (Product #R6504), Basal Medium Eagle (BME), Ham's, and Minimum Essential Medium Eagle (MEM, or EMEM), which contains amino acids, salts (potassium chloride, magnesium sulfate, sodium chloride, and sodium dihydrogen phosphate), glucose and vitamins (folic acid, nicotinamide, riboflavin, B-12). Cells are cultured overnight at 5% C02 at 37 degrees Celsius in a fully humidified incubator. After overnight culture, cells are examined under an inverted light microscope. To collect adherent cells, media from the Petri dish is decanted and 10 ml of PBS was added to the Petri dish.
  • RPMI- 1640 Roswell Park Memorial Institute Media
  • BME Basal Medium Eagle
  • MEM Minimum Essential Medium Eagle
  • EMEM Minimum Essential Medium Eagle
  • Cells are cultured overnight at 5% C02 at 37 degrees Celsius in a fully humidified incubator. After overnight
  • the Petri dish is gently rocked back and forth 5 times and PBS was then removed with a pipette, with care being taken not to disrupt adherence cells. This procedure is repeated a second time. Subsequently all PBS is removed and 2 ml of Trypsin-EDTA solution (Sigma Aldrich, St Louis, catalogue #T3924) is added to cover the surface area of the Petri dish. The Petri dish is subsequently placed into an incubator at 37 Celsius for 2 minutes. Cells are then detached by gentle flushing of PBS over the Petri dish. Cells in 10 ml of PBS are centrifuged for 10 minutes at 400 g.
  • the cell pellet is respuspended by tapping gentle against a hard surface 5 times and subsequently complete 5 ml of complete DMEM is added to the resuspended pellet.
  • Cells are counted and 1. times.10.sup.5 cells were placed in a T75 flask (Fisher Scientific, Portsmouth N.H.) containing 15 ml of media and cultured in a fully humidified incubator at 37 Celsius, 5% C02. The cells are then subcultured and passaged twice a week. Passaging involved trypsinization of cells when they reach approximately 75% confluence. After trypsinization and washing 1. times.10.sup.5 cells are placed into T75 flasks in 15 ml complete DMEM. Based on these conditions cells are typically passaged 2 times a week.
  • T75 flask Fisher Scientific, Portsmouth N.H.
  • donors are mobilized by administration of G-CSF (filgrastim: neupogen) at a concentration of lOug/kg/day by subcutaneous injection for 2-7 days, more preferably 4-5 days.
  • G-CSF filamentgrastim: neupogen
  • Peripheral blood mononuclear cells are collected using an apheresis device such as the AS 104 cell separator (Fresenius Medical). 1-40 x 109 mononuclear cells are collected, concentrated and injected into the peritoneaum in a localized manner through the use of a biodegradable polymer.
  • Variations of this procedure may include steps such as subsequent culture of cells to enrich for various populations known to possess angiogenic and/or neurogenic, and/or anti-atrophy Additionally cells may be purified for specific subtypes before and/or after culture. Treatments can be made to the cells during culture or at specific timepoints during ex vivo culture but before infusion in order to generate and/or expand specific subtypes and/or functional properties.
  • mesenchymal cells are generated through culture.
  • US patent # 5,486,359 describes methods for culturing such and expanding mesenchymal stem cells, as well as providing antibodies for use in detection and isolation.
  • US patent # 5,942,225 teaches culture techniques and additives for differentiation of such stem cells which can be used in the context of the present invention to produce increased numbers of cells with angiogenic capability.
  • US patent # 6,387,369 teaches use of mesenchymal stem cells for regeneration of cardiac tissue, we believe that in accordance with published literature [37, 38] stem cells generated through these means are actually angiogenic ally potent and therefore may be utilized in the context of the current invention for treatment/amelioration of surgical adhesions.
  • mesenchymal stem cells induce angiogenesis through production of factors such as vascular endothelial growth factor, hepatocyte growth factor, adrenomedullin, and insulin-like growth factor- 1 [39].
  • Mesenchymal stem cells are classically obtained from bone marrow sources for clinical use, although this source may have disadvantages because of the invasiveness of the donation procedure and the reported decline in number of bone marrow derived mesenchymal stem cells during aging.
  • Alternative sources of mesenchymal stem cells include adipose tissue [40], placenta [22, 41], scalp tissue [42] and cord blood [43].
  • a recent study compared mesenchymal stem cells from bone marrow, cord blood and adipose tissue in terms of colony formation activity, expansion potential and immunophenotype. It was demonstrated that all three sources produced mesenchymal stem cells with similar morphology and phenotype.
  • autologous mesenchymal stem cells are available in the form of adipocyte- derived cells, it will be useful to utilize this source instead of allogeneic cord-blood derived cells.
  • cord blood derived mesenchymal stem cells may be more advantageous for use in situations where autologous cells are not available, and expansion is sought.
  • mesenchymal stem cells from the cord blood to expand in vitro also allows the possibility of genetically modifying these cells in order to: a) decrease immunogeneicity; b) enhance angiogenic potential; and c) augment survival following administration.
  • ex vivo manipulation is applicable to all cell types described in the current application.
  • cells may be transfected using immune suppressive agents. Said agents include soluble factors, membrane-bound factors, and enzymes capable of causing localized immune suppression.
  • soluble immune suppressive factors include: IL-4 [45], IL-10 [46], IL-13 [47], TGF-b [48], soluble TNF-receptor [49], and IL-1 receptor agonist [50].
  • Membrane- bound immunoinhibitor molecules that may be transfected into stem cells for use in practicing the current invention include: HLA-G [51], FasL [52], PD-IL [53], Decay Accelerating Factor [54], and membrane-associated TGF-b [55].
  • Enzymes which may be transfected in order to cause localized immune suppression include indolamine 2,3 dioxygenase [56] and arginase type II [57].
  • mesenchymal stem cells may be transfected with genes such as VEGF[58], FGF1 [59], FGF2 [60], FGF4 [61], FrzA [62], and angiopoietin [63].
  • Ability to induce angiogenesis may be assessed in vitro prior to administration of said transfected cells in vivo.
  • Methods of assessing in vitro angiogenesis stimulating ability are well known in the art and include measuring proliferation of human umbilical vein derived endothelial cells. Since one of the problems of cell therapy in general is viability of the infused cells subsequent to administration, it may be desired in some forms of the invention to transfect mesenchymal cells with genes protecting said cells from apoptosis. Anti-apoptotic genes suitable for transfection may include bcl-2 [64], bcl-xl [65], and members of the XIAP family [66]. Alternatively it may be desired to increase the proliferative lifespan of said mesenchymal stem cells through transfection with enzymes associated with anti-senescence activity. Said enzymes may include telomerase or histone deacetylases. The various embodiments of the invention described above for cord blood and embryonic stem cells can also be applied for mesenchymal stem cells.
  • Adipose derived stem cells express markers such as CD9; CD29 (integrin beta 1); CD44 (hyaluronate receptor); CD49d,e (integrin alpha 4, 5); CD55 (decay accelerating factor); CD 105 (endoglin); CD 106 (VCAM-1); CD 166 (ALCAM). These markers are useful not only for identification but may be used as a means of positive selection, before and/or after culture in order to increase purity of the desired cell population. In terms of purification and isolation, devices are known to those skilled in the art for rapid extraction and purification of cells adipose tissues.
  • US patent #6,316,247 describes a device which purifies mononuclear adipose derived stem cells in an enclosed environment without the need for setting up a GMP/GTP cell processing laboratory so that patients may be treated in a wide variety of settings.
  • One embodiment of the invention involves attaining 10-200 ml of raw lipoaspirate, washing said lipoaspirate in phosphate buffered saline, digesting said lipoaspirate with 0.075% collagenase type I for 30-60 min at 37°C with gentle agitation, neutralizing said collagenase with DMEM or other medium containing autologous serum, preferably at a concentration of 10% v/v, centrifuging the treated lipoaspirate at approximately 700-2000g for 5-15 minutes, followed by resuspension of said cells in an appropriate medium such as DMEM. Cells are subsequently filtered using a cell strainer, for example a 100 ⁇ nylon cell strainer in order to remove debris.
  • a cell strainer for example a 100 ⁇ nylon cell strainer in order to remove debris.
  • Filtered cells are subsequently centrifuged again at approximately 700-2000g for 5-15 minutes and resuspended at a concentration of approximately Ixl06/cm2 into culture flasks or similar vessels. After 10-20 hours of culture non-adherent cells are removed by washing with PBS and remaining cells are cultured at similar conditions as described above for culture of cord blood derived mesenchymal stem cells. Upon reaching a concentration desired for clinical use, cells are harvested, assessed for purity and administered in a patient in need thereof as described above.
  • the various embodiments of the invention described above for cord blood and embryonic stem cells can also be applied for adipose derived stem cells.
  • the bulge area of the hair follicle bulge is an easily accessible source of pluripotent mesenchymal-like stem cells.
  • One embodiment of the current invention is the use of hair follicle stem cells for treatment of surgical adhesions or their prevention. Said cells may be used therapeutically once freshly isolated, or may be purified for particular sub-populations, or may be expanded ex vivo prior to use. Purification of hair follicle stem cells may be performed from cadavers, from healthy volunteers, or from patients undergoing plastic surgery. Upon extraction, scalp specimens are rinsed in a wash solution such as phosphate buffered saline or Hanks and cut into sections 0.2-0.8 cm.
  • a wash solution such as phosphate buffered saline or Hanks
  • Subcutaneous tissue is de-aggregated into a single cell suspension by use of enzymes such as dispase and/or collagenase.
  • scalp samples are incubated with 0.5% dispase for a period of 15 hours.
  • the dermal sheath is further enzymatically de-aggregated with enzymes such as collagenase D.
  • Digestion of the stalk of the dermal papilla the source of stem cells is confirmed by visual microscopy.
  • Single cell suspensions are then treated with media containing fetal calf serum, and concentrated by pelletting using centrifugation. Cells may be further purified for expression of markers such as CD34, which are associated with enhanced proliferative ability.
  • collected hair follicle stem cells are induced to differentiate in vitro into neural-like cells through culture in media containing factors such as FGF-1 , FGF-2, NGF, neurotrophin-2, and/or BDNF. Confirmation of neural differentiation may be performed by assessment of markers such as Muhashi, polysialyated N-CAM, N- CAM, A2B5, nestin, vimentin glutamate, synaptophysin, glutamic acid decarboxylase, serotonin , tyrosine hydroxylase, and GABA. Said neuronal cells may be administered systemically, or locally in a patient with ED.
  • the various embodiments of the invention described above for cord blood and embryonic stem cells can also be applied for hair follicle stem cells.
  • Parthenogenically derived stem cells can be generated by addition of a calcium flux inducing agent to activate oocytes, followed by purifying and expanding cells expressing embryonic stem cell markers such as SSEA-4, TRA 1-60 and/or TRA 1- 81.
  • Said parthenogenically derived stem cells are totipotent and can be used in a manner similar to that described for embryonic stem cells in the practice of the current invention.
  • the various embodiments of the invention described above for cord blood and embryonic stem cells can also be applied for parthenogenically derived stem cells. Reprogramming of non-stem cells to endow them with stem cell characteristics can generate stem cells for use in the practice of the current invention.
  • reprogramming cells are that ability to withdraw autologous cells, which may have limited stem cell potential, endow said autologous cells with stem cell, or stem cell-like, properties, and reintroduce said autologous cells into the patient.
  • the various embodiments of the invention described above for cord blood and embryonic stem cells can also be applied for reprogrammed stem cells.
  • tissue derived side population cells may be utilized either freshly isolated, sorted into subpopulations, or subsequent to ex vivo culture, for the treatment and/or prevention of surgical adhesions.
  • side population cells may be derived from tissues such as pancreatic tissue, liver tissue, smooth muscle tissue, striated muscle tissue, cardiac muscle tissue, bone tissue, bone marrow tissue, bone spongy tissue, cartilage tissue, liver tissue, pancreas tissue, pancreatic ductal tissue, spleen tissue, thymus 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. More optimally, side population cells obtained from smooth muscle tissue are administered at a concentration and frequency sufficient to induce a prophylactic effect on surgical adhesion formation.
  • stem cell sources may be utilized as regenerative cells useful for inhibition of development of fibrosis or peritoneal adhesions. Protocols for the clinical-grade generation of said stem cell sources may be found in the literature and are incorporated here by reference. Specifically, bone marrow mesenchymal [67-71], placental [72], cord blood [73], and bone marrow mononuclear [74, 75], stem cell sources are all described in the references incorporated herein. In some embodiments cells are first treated with hypoxia to increase therapeutic activity as described [76].
  • the current invention seeks to provide a method of prevention of surgical adhesion formation using cell populations such as monocyte or stromal vascular fraction cells for stimulation of regenerative/anti-inflammatory and antifibrotic processes.
  • cell populations such as monocyte or stromal vascular fraction cells for stimulation of regenerative/anti-inflammatory and antifibrotic processes.
  • One of the central features of the invention is the use of monocytes that have been manipulated extracorporally to induce a regenerative/antiifibrotic effect. Said monocytes may be used in an autologous, allogeneic, or xenogeneic manner. Monocyte activity may be modulated by treatment with chemical agents, a particular class of which is modulators of the peroxisome proliferator-activated receptors (PPARs).
  • PPARs peroxisome proliferator-activated receptors
  • This receptor family acts as a ligand-activated nuclear transcription factors that belong to the steroid/thyroid receptor superfamily, and is classified as three subtypes, PPARa, ⁇ and PPARy [77, 78]. They can be activated by natural (fatty acids) and synthetic ligands, and exhibit different functions.
  • PPARa is responsible for regulating lipid metabolism, lipoprotein formation and transport; PPAR controls lipid oxidation, and PPARy promotes adipogenesis and enhances pre-adipocyte differentiation into adipocyte [79].
  • each of these receptors have been shown to exert other activities in different tissues. PPARy and PPARa are significantly involved in preventing inflammation in the immune system [80].
  • T cells have been demonstrated to contribute to formation of surgical adhesions through the production of Thl cytokines [81, 82].
  • inhibitors of T cell function such as PD-1 activation have been demonstrated to inhibit formation of peritoneal adhesions [83]. Accordingly, within the practice of the current invention, the use of inhibitors of inflammation and/or Thl function is utilized for the inhibition of surgical adhesion formation together with regenerative cells.
  • PPARy exerts its anti-inflammatory effects mainly by downregulating expression of proinflammatory mediators such as TNFa, IL- 1 ⁇ , and IL-6, etc. and upregulating expression of anti-inflammatory factors such as IL-10, CYP4A, and Catalase [84]. It has also been found that PPARy enhances the alternative M2 macrophage phenotype and promotes human monocyte into M2 differentiation leading to a more pronounced anti-inflammatory activity on Ml macrophages [85, 86]. The current invention teaches the use of PPARy activators to generate monocyte/macrophage populations that are suitable for use in prevention and/or treatment of surgical adhesions.
  • PPARy agonists are used because of selectivity towards the monocytic lineage.
  • PPARa expresses in all the same cells as PPARy with exception of dendritic cells and alveolar macrophages [79], PPARa controls inflammation in hepatocytes [80], inhibits airway inflammation induced by lipopolysaccharide [87] and inflammatory ear-swelling response to leukotriene B4 [88], and mediates the acute anti-inflammatory effects of the anticholesterolemic drug simvastatin [89].
  • PPARy agonists include the cyclopentenone prostaglandin 15-deoxy- A12, 14-prostaglandin J2 (15d-PGJ2), 15-hydroxyeicosatetraenoic acid (15-HETE) and 13-hydroxyoctadecadienoic acid (13-HODE) and synthetic agonists such as thiazolidinediones (TZDs)/ rosiglitazone (Avandia), pioglitazone (Actos) and Troglitazone (Rezulin) [84].
  • PPARy agonists include the cyclopentenone prostaglandin 15-deoxy- A12, 14-prostaglandin J2 (15d-PGJ2), 15-hydroxyeicosatetraenoic acid (15-HETE) and 13-hydroxyoctadecadienoic acid (13-HODE) and synthetic agonists such as thiazolidinediones (TZDs)/ rosiglitazone (Avan
  • NSAIDs non-steroidal anti- inflammatory drugs
  • Other drugs include Ascochlorin and its derivatives such as 4-O-carboxymethyl ascochlorin are also found to be potent PPARy agonists [90].
  • TZDs are used in patients with type II diabetes in the clinic [91, 92], recent studies have increasingly demonstrated the therapeutic effects of PPARy ligands reach far beyond their use as insulin sensitizers in terms of treating other inflammatory indications.
  • Some compounds such as ragaglitazar, GW-409544, KRP-297 can activate both PPARy and PPARa [84], called dual PPARo/PPARy ligands .
  • Another compound PD168 is found to be a potent PPARy agonist with weak activity at PPARa.
  • a patient prior to surgery undergoes a liposuction to collect stromal vascular fraction (SVF) cells.
  • This population is known to contain a combination of alternatively activated macrophages, T regulatory cells, and mesenchymal stem cells [93].
  • T regulatory cells a T cell that stimulates apoptosis
  • mesenchymal stem cells a T cell that stimulates apoptosis.
  • mesenchymal stem cells a liposuction to collect stromal vascular fraction (SVF) cells.
  • T regulatory cells a combination of alternatively activated macrophages, T regulatory cells, and mesenchymal stem cells [93].
  • mesenchymal stem cells mesenchymal stem cells
  • activated macrophages possess anti-inflammatory effects [94] and may be useful in suppressing macrophage hyperreactivity found in fibrotic patients that contributes to pathology [95-98].
  • T regulatory cells inhibit autoreactive T cells [99], as well as polarize macrophages towards an anti-inflammatory state [100].
  • Mesenchymal stem cells not only have the potential to immune modulate through induction of T regulatory cells [101-103], suppression of macrophage activation [104] and release of anti-inflammatory factors [105], but also have ability to induce regeneration of damaged alveolar tissue [106].
  • therapeutic activities of SVF to prevent formation of surgical adheasions are enhanced by exposure to a PPAR agonist, more preferable to an agonist of PPAR-gamma.
  • Said agonists are known in the literature and described in the previous section.
  • autologous SVF cells are exposed to a concentration of 100 micro Moles of rosiglitazone for a period of lhr-1000 hours, more preferably approximately 24 hours to augment anti-inflammatory activities.
  • the patient is administered a systemic concentration of a PPAR- gamma agonist together with autologous SVF.
  • Administration of SVF may be local in the peritoneal cavity or systemic.
  • administration of the SVF is performed via a biodegradable polymer.
  • Bone marrow transplantation has been clinically used for hematopoietic reconstitution for more than 40 years, thus procedures for bone marrow extraction and manipulation are routine.
  • procedures for bone marrow extraction and manipulation are routine.
  • previous researchers have demonstrated feasibility of administering autologous bone marrow to prevent other fibrotic conditions [107]. Therefore it is within the scope of the current invention to utilize autologous bone marrow mononuclear alone or after generation of mesenchymal stem cells thereof. Protocols for extraction of bone marrow include the following:
  • G- CSF granulocyte colony-stimulating factor
  • Bone marrow may be collected by a total of 20 (10 on each iliac crest) punctures was made, always in different parts, with a Jamshidi needle. Approximately, 10 mL of bone marrow is aspirated from each puncture, using a 20-mL syringe previously filled with 0.5 mL of heparin sodium (5000 IU/mL). The collected material is added to a BaxterTM (Baxter Healthcare Corporation) collection bag. The clamps of the collection kit are opened and the bone marrow total content is filtered by gravity. The collection bag packed is then placed in a sterile field and opened inside a laminar flow hood. The collection bag content is collected with the aid of a coupler, in strictly aseptic conditions.
  • Ficoll-Hypaque PremiumTM is placed in polypropylene centrifuge tubes of 50 mL and then 30 mL of bone marrow (diluted 1 : 1 with saline) is added, to give a total a volume of 50 mL inside the tube with the Ficoll-Hypaque Premium solution. Afterward, the tubes are centrifuged at 300 g for 30 min at 18°C. The ring of mononuclear cells formed above the Ficoll layer is collected with the aid of a sterile Pasteur pipette. Then, the mononuclear cells are transferred to other 50 mL centrifuge tubes. The volume is completed with RPMI culture medium.
  • the total volume is filtered in a Cell StrainerTM with a porosity of 100 ⁇ to remove any impurities, cellular aggregates, and/or bone fragments.
  • Two counts with Turk's solution and trypan blue are performed: the first count after the collection of bone marrow and the second count after the processing of the cells.
  • the infusions into the patients prior to surgery are then made into a (medial brachial) peripheral vein, promptly after the preparation, separation, and counting of the mononuclear cells.
  • the mononuclear cells (BMMC) pool solution, diluted in physiological serum at 5% albumin (ASS) to a final volume of 30 mL, are slowly infused, over ⁇ 20 min.
  • cells may be first treated by laser irradiation to enhance antifibrotic properties.
  • Laser irradiation may be performed on the cells prior to administration, and/or the patient may be exposed to laser irradiation on the peritoneal areas proximal to the site of surgically induced injury. Choice of laser and duration is based on patient disease state and inflammatory markers.
  • laser administration is performed as previously described [108].
  • Cells may be administered directly into the peritoneal cavity, or in the form of a biodegradable matrix. In some embodiments cells may be placed together with Seprafilm.
  • various delivery agents may be utilized, examples of which include polyacrylic acid (carbopol 974-P NF) or cross-linked polyacrylic acid like, for example, carbomer or polycarbophyl, or other derivatives of polyacrylic acids like, for example, polymethylmethacrylates; cellulose or derivatives of cellulose, like, for example, sodium carboxymethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose; other polysaccharides, like, for example, chitosan and its pharmaceutically acceptable salts, like, for example, chitosan glutamate, or, furthermore, polysaccharides like alginates, pregelatinised starches, pectins; glycosoaminoglycans, like, for example, hyaluronic acid, chondroitin sulphate; polyvinylpyrrolidone; gelatine; or mixtures of the mentioned agents.
  • polyacrylic acid carbomer or polycarbophyl
  • polymethylmethacrylates
  • cells may be sprayed on utilizing a thrombin fibrinogen mixture.
  • various growth factors may be added to the composition containing cells in order to extent viability/therapeutic activity of cells, said growth factors may be selected from a group of growth factors including: platelet-derived growth factor (PDGF), platelet- derived angiogenesis factor (PDAF), vascular endothelial growth factor (VEGF), platelet- derived epidermal growth factor (PDEGF), platelet factor 4 (PF-4), transforming growth factor .beta.
  • PDGF platelet-derived growth factor
  • PDAF platelet- derived angiogenesis factor
  • VEGF vascular endothelial growth factor
  • PEGF platelet- derived epidermal growth factor
  • PF-4 platelet factor 4
  • TGF-.beta. acidic fibroblast growth factor (FGF-A), basic fibroblast growth factor (FGF-B, insulin-like growth factors 1 and 2 (IGF-1 and IGF-2), .beta, thromboglobulin-related proteins (.beta. TG), thrombospondin (TSP), fibronectin, von Wallebrand factor (vWF), fibropeptide A, fibrinogen, albumin, plasminogen activator inhibitor 1 (PAI-1), osteonectin, regulated upon activation normal T cell expressed and presumably secreted (RANTES), gro-.
  • FGF-A acidic fibroblast growth factor
  • FGF-B basic fibroblast growth factor
  • IGF-1 and IGF-2 insulin-like growth factors 1 and 2
  • .beta thromboglobulin-related proteins
  • TSP thrombospondin
  • fibronectin von Wallebrand factor
  • fibropeptide A fibrinogen, albumin, plasminogen activator inhibitor 1 (
  • One of skilled in the art may, without extensive experimentation, utilize various in vitro assays to screen for optimized combinations of growth factors with cells administered.
  • the use of bone marrow for prevention of surgical adhesions relates to the need in this patient population for angiogenesis in part to reduce tissue scarring, and in part to allow for regeneration of the mesothelium.
  • the use of bone marrow for the stimulation of angiogenesis and reduction of scarring has been previously described in other conditions but not in the prevention of surgical adhesions, and the artisan skilled in the art is referred to the examples below for reference in the practice of the invention as relates to prevention of surgical adhesions.
  • the condition of limb ischemia also referred here to as Critical Limb Ischemia (CLI) is provided as a source of materials and clinical grade preparation of cells, so that the skilled artisan may apply these experiences for the practice of the invention.
  • CLI Critical Limb Ischemia
  • Tateishi-Yuyama et al [109] used the technique of bone marrow extraction and mononuclear cell preparation to generate an autologous source of stem cells for stimulation of angiogenesis in ischemic limb treatment.
  • mononuclear cells at concentrations ranging between 0.7-2.8 x 109 cells in the gastrocnemius muscle of the ischemic leg using approximately 40 injections in a volumes of 0.75 ml.
  • Statistically significant increases in ankle brachial index, trancutaneous oxygen pressure, pain free walking time, and amelioration of rest pain were detected at 4 and 24-week follow-up.
  • the investigators also demonstrated production of proangiogenic cytokines by the injected cells.
  • Nizankowski et al treated 10 CLI patients with condensed bone marrow stem cells and observed improvement using laser doppler flux and percutaneous oxygen partial pressure, as well as decrease in pain severity. Interestingly no correlation was found between cell number injected and effect [110].
  • Durdu et al performed intramuscular injection of autologous bone marrow mononuclear cells to 28 CLI patients who were nonresponders to conservative therapy and ineligible for revascularization. Of the 28 patients, only 1 needed amputation. Statistically significant increases in rest pain scores, peak walking time, and quality of life were noted. Angiography evidence of collateral vessel formation was observed in 22 of the patients at 6 months [111].
  • adipose stromal vascular fraction (SVF) cells are used as a regenerative cell population.
  • the mononuclear fraction of adipose tissue referred to as the stromal vascular fraction (SVF)
  • the cells comprising SVF morphologically resemble fibroblasts and were demonstrated to differentiate into pre-adipocytes and functional adipose tissue in vitro [124].
  • MSC Mesenchymal stem cells
  • markers such as CD90, CD105, and CD73, while lacking expression of CD14, CD34, and CD45, and being able to differentiate into adipocytes, chondrocytes, and osteocytes in vitro after treatment with differentiation inducing agents [128].
  • MSC Mesenchymal stem cells
  • xenogenic cells of the mesenchymal lineage may be utilized in combinations described herein for the treatment or prevention of surgical adhesions.
  • Adipose tissue has attracted interest as a possible alternative to bone marrow as a source for stem cell extraction. Enticing characteristics of adipose derived cells include: a) ease of extraction, b) higher content of mesenchymal stem cells (MSC) as compared to bone marrow, and c) ex vivo expandability of MSC is approximately equivalent, if not superior to bone marrow [139].
  • MSC immune modulatory/anti- inflammatory/antifibrotic properties
  • Allogeneic bone marrow derived MSC have been used by academic investigators with clinical benefit treatment of diseases such as graft versus host (GVHD) [160-165], osteogenesis imperfecta [166], Hurler syndrome, metachromatic leukodystrophy [167], and acceleration of hematopoietic stem cell engraftment [168-170].
  • GVHD graft versus host
  • 166 osteogenesis imperfecta
  • Hurler syndrome metachromatic leukodystrophy
  • 167 metachromatic leukodystrophy
  • hematopoietic stem cell engraftment hematopoietic stem cell engraftment
  • Angioblast Systems has recently announced initiation of Phase II trials using Mesenchymal Precursor Cells for stimulation of cardiac angiogenesis [173].
  • Neuronyx is in Phase I clinical trials using allogeneic human adult bone marrow-derived somatic cells (hABM-SC) for post infarct healing [174].
  • hABM-SC allogeneic human adult bone marrow-derived somatic cells
  • the company Cytori is currently conducting two European clinical trials using autologous adipose-derived mononuclear cells, of which MSC are believed to be the therapeutic population [175].
  • the PRECISE trial is a 36-patient safety and feasibility study in Europe evaluating adipose-derived stem and regenerative cells as a treatment for chronic cardiac ischemia.
  • the APOLLO trial is a 48-patient safety and feasibility study in Europe to evaluate adipose-derived regenerative cells as a treatment for heart attacks [176].
  • Allogeneic uses of adipose derived MSC included treatment of GVHD associated liver failure [144], steroid refractory GVHD [145, 177], Allogeneic placenta and cord blood-derived MSC have also been used for treatment of heart failure [178] and Buerger's Disease [179], respectively. From the above mentioned clinical trials of allogeneic MSC, graft versus host or pathological immunological reactions have not been reported.
  • adipocytes were capable of inducing TNF- alpha secretion from macrophage cell lines in vitro [182].
  • Clinical studies demonstrated that adipocytes also directly release a constitutive amount of TNF-alpha and leptin, which are capable of inducing macrophage secretion of inflammatory mediators [183].
  • monocytes/macrophages are isolated from adipose tissue, they in fact possess antiinflammatory functions characterized by high expression of IL-10 and IL-1 receptor antagonist [158, 184, 185].
  • M2 adipose derived macrophages
  • adipose derived macrophages have an "M2" phenotype, which physiologically is seen in conditions of immune suppression such as in tumors [186], post-sepsis compensatory anti-inflammatory syndrome [187, 188], or pregnancy associated decidual macrophages [189].
  • M2 monocytic/macrophage compartment of the SVF
  • administrations of ex vivo generated M2 macrophages have been demonstrated to inhibit kidney injury in an adriamycin-induced model [191].
  • M2-like microglial cells are believed to inhibit progression in the EAE model [192].
  • the anti-inflammatory activities of M2 cells is a potential mechanism of therapeutic effect of SVF cells when isolated from primary sources and not expanded.
  • cells with regenerative activity are aerosolized and administered directly into the peritoneal cavity or localized at the site of surgical injury.
  • conditioned media derived from said cells with regenerative activity are aerosolized and delivered either directly into the peritoneal cavity or at the place of surgical injury.
  • the infraumbilical region is infiltrated with 0.5% Xylocaine with 1 :200,000 epinephrine. After allowing 10 minutes for hemostasis, a 4mm cannula attached to a 60cc Toomey syringe is used to aspirate 500 cc of adipose tissue in a circumincisional radiating technique. Based on laboratory experience a volume of 500 cc's should provide nearly 1.5-2 xl08 SVF cells, more than our desired goal of 1.14 x 108 SVF cells.
  • each of 9 syringes As each of 9 syringes are filled, it will be removed from the cannula, capped, and exchanged for a fresh syringe in a sterile manner within the sterile field.
  • the aspirated adipose tissue remains in the 60 cc syringes and is labeled with the patient's initials, medical record number, and content.
  • the adipose filled syringes is then transported to the processing lab.
  • the syringe-filled lipoaspirate is then placed into two sterile 500 mL centrifuge containers and washed three times with sterile Dulbecco's phosphate -buffered saline to eliminate erythrocytes.
  • ClyZyme/PBS (7mL/500mL) is added to the washed lipoaspirate using a 1 : 1 volume ratio.
  • the centrifuge containers are then sealed and placed in a 37° C shaking water bath for one hour then centrifuged for 5 min at 300rcf. During centrifugation, the stromal cells form a pellet in the bottom of the container while the adipocyte layer and debris remains suspended.
  • the stromal cells are resuspended within Isolyte in separate sterile 50 mL centrifuge tubes. The tubes are then centrifuged for 5 min. at 300rcf and the Isolyte is removed, leaving cell pellets. The pellets are then resuspended in 40 ml of Isolyte, centrifuged again for 5 min at 300rcf. The supernatant again is removed. The cell pellets are then combined filtered through 100 m cell strainers into a sterile 50ml centrifuge tube and centrifuged for 5 min at 300rcf and the supernatant removed, leaving the pelleted adipose stromal cells.
  • the cells are then be counted with a hemacytometer and cell concentration will be adjusted to 7x106 cells/ml. Aliquots of cell suspension are set aside for anaerobic and aerobic bacterial and fungal sterility testing and endotoxin testing. The SVF fraction will only be released for injection after receiving negative sterility test results and the endotoxin assay results of level of EU less than or equal to 5 EU/kg/hr.
  • a therapeutic composition for inhibiting development of fibrosis in a patient in need of such treatment, the therapeutic composition being biodegradable and implantable at, near or in the area of risk for development of fibrotic tissue, the therapeutic composition comprising an antifibrotic agent to degrade, shrink, relax, or stretch at least a portion of the fibrotic tissue and being configured to immediately release an effective amount of the antifibrotic agent within 24 hours.
  • Said antifibrotic agent may be a cell, an enzyme, a small molecule, a nucleic acid composition capable of inducing RNA interference targeting a gene associated with fibrosis, or a gene therapy.
  • Antifibrotic agents useful for the practice of the invention include elastase, elastase-2a, elastase-2b, neutrophil elastase, proteinase-3, endogenous vascular elastase, cathepsin G, mast cell chymase, mast cell tryptase, plasmin, thrombin, granzyme B, cathepsin S, cathepsin K, cathepsin L, cathepsin B, cathespin C, cathepsin H, cathespin F, cathepsin G, cathepsin O, cathepsin R, cathepsin V (cathepsin 12), cathepsin W, calpin 1, calpin 2, chondroitinase ABC, chondroitinase AC, hyaluronidase, chymopapain, chymotrypsin, legumain, cathepsin Z (cat
  • the therapeutic composition comprises a drug depot having an immediate release component that when it contacts an activator (e.g., liquid or semi-solid activator, energy, etc.), the activator causes the drug depot to release an effective amount of the antifibrotic agent together with a cellular composition capable of inducing antifibrotic effects.
  • an activator e.g., liquid or semi-solid activator, energy, etc.
  • a therapeutic composition for treating fibrous tissue in a patient in need of such treatment, said therapeutic composition possessing biodegradable properties and implantable at, near, or in a fibrous tissue, said therapeutic composition comprising an antifibrotic agent to degrade, shrink, relax or stretch at least a portion of the fibrous tissue and being configured to immediately release an effective amount of the antifibrotic agent within 24 hours and configured to provide sustained release of the antifibrotic agent over a period of up to one year to treat the fibrous tissue.
  • Said therapeutic composition can treat normal and/or abnormal fibrous tissue.
  • Said antifibrotic utilized may be the same combinations described earlier in the specification. Specifically, antifibrotic agents are chosen based on ability to enzymatically degrade or shrink the fibrotic tissue.
  • the antifibrotic agent when it is an enzyme, it will reduce pressure by degrading proteoglycans (PG) so that PGs are not available to hold water.
  • the antifibrotic agent can be a protease or glycanase, which is not proteolytic, alternatively genes encoding said enzymes may be administered in order to overcome pharmacological/immunological limitations of protein expression.
  • the antifibrotic agent instead of an enzyme, can be an agent that dehydrates the fibrotic tissue, such as a polycationic polymer.
  • the anti-fibrotic agent may be a hormone, such as for example, relaxin, which inhibits collagen production and stimulates collagen degradation.
  • the anti-fibrotic agent may be a cytokine, drug, cell, or nucleic-acid-based material that modulates the function, viability, or proliferation of fibroblasts or other cells in the fibrotic tissue.
  • the antifibrotic agent may be cells that inhibit collagen production and/or stimulates collagen degradation.
  • Cells include stem cells but also include non-stem cells such as monocytes, mononuclear cells obtained from peripheral blood, or platelet rich plasma.
  • a therapeutic composition is administered to a patient in need containing a cell population, an antioxidant, an antifibrotic agent, and an anti-inflammatory.
  • Numerous anti-inflammatories are known in the art and include statins such as atorvastatin, simvastatin, pravastatin, cerivastatin, mevastatin, velostatin, fluvastatin, lovastatin, rosuvastatin and fluindostatin (Sandoz XU-62-320), dalvastain, eptastatin, pitavastatin, or pharmaceutically acceptable salts thereof or a combination thereof, sulindac, flufenamic acid, clonixeril, clonixin, meclofenamic acid, flunixin, colchicine, demecolcine, allopurinol, ketoprofen, aclofenac, aloxiprin, aproxen, aspirin, diflunisal, fenopro
  • statins
  • thalidomide analogues which reduce TNF-. alpha, production by macrophages
  • bone morphogenetic protein (BMP) type 2 or BMP-4 inhibitors of caspase 8, a TNF-. alpha, activator
  • quinapril an inhibitor of angiotensin II, which upregulates TNF-. alpha.
  • interferons such as IL-11 (which modulate TNF-. alpha, receptor expression), and aurin-tricarboxylic acid (which inhibits TNF-.
  • Bossolasco P., et al., Molecular and phenotypic characterization of human amniotic fluid cells and their differentiation potential. Cell Res, 2006.16(4): p. 329-36.
  • TGF-beta influences the life and death decisions of T lymphocytes. Cytokine Growth Factor Rev, 2000. 11(1-2): p. 71-9. Mellor, A.L. and D.H. Munn, IDO expression by dendritic cells: tolerance and tryptophan catabolism. Nat Rev Immunol, 2004. 4(10): p. 762-74.
  • angiogenesis factors new insights into their mechanism of action. Exs, 1997. 79: p. 159-92.
  • Ad5FGF-4 adenovirus 5 fibroblast growth factor-4
  • statins 89. Paumelle, R., et al., Acute antiinflammatory properties of statins involve
  • Prockop, D.J. Marrow stromal cells as stem cells for nonhematopoietic tissues.
  • mesenchymal stem cells have immunomodulatory capacities. Stem Cells Dev, 2007. 16(4): p. 597-604. 132. Chao, K.C., et al., Islet-like clusters derived from mesenchymal stem cells in Wharton's Jelly of the human umbilical cord for transplantation to control type 1 diabetes. PLoS ONE, 2008. 3(1): p. el451.
  • Kern, S., et al. Comparative analysis of mesenchymal stem cells from bone
  • MID metachromatic leukodystrophy
  • MPS-IH Hurler syndrome
  • CNS-derived interleukin-4 is essential for the regulation of autoimmune inflammation and induces a state of alternative activation in microglial cells. J Neurosci, 2007. 27(40): p. 10714-21.

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Abstract

L'invention concerne des procédés de prévention de développement et/ou de traitement d'une fibrose de tissu provoquée, par exemple, par des adhérences chirurgicales au moyen de l'administration de cellules souches et de cellules souches en combinaison avec des matrices ou d'autres molécules, ou des produits dérivés de celles-ci. Dans un mode de réalisation, des cellules souches pluripotentes sont disposées par voie intrapéritonéale seules, ou fixées à une matrice ou incorporées dans celle-ci. Diverses combinaisons de cellules, de produits de celles-ci et de facteurs de régénération sont capables de réduire la formation d'adhérences chirurgicales à la fois prophylactiquement et thérapeutiquement.
PCT/US2014/014733 2013-02-05 2014-02-04 Prévention et traitement de fibrose de tissu WO2014123930A2 (fr)

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Cited By (6)

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CN105018428A (zh) * 2015-05-27 2015-11-04 贵州北科泛特尔生物科技有限公司 脐带血造血干细胞的体外扩增方法
CN105670987A (zh) * 2016-03-21 2016-06-15 杭州市萧山区中医院 一种毛囊干细胞诱导分化为血管内皮细胞的抑制方法
CN108441474A (zh) * 2018-03-23 2018-08-24 天晴干细胞股份有限公司 一种来源于脐带血、胎盘造血干细胞分离纯化的方法
CN109364090A (zh) * 2018-12-06 2019-02-22 西华大学 油橄榄叶提取物在制备防治胎儿酒精综合症药物中的应用
KR20190026772A (ko) * 2016-07-01 2019-03-13 고쿠리츠다이가쿠호진 도호쿠다이가쿠 장기 섬유증의 예방 또는 치료제
WO2020146874A1 (fr) * 2019-01-11 2020-07-16 Figene, Llc Cellules fibroblastes régénératrices

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* Cited by examiner, † Cited by third party
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DK1218489T3 (da) * 1999-09-24 2009-06-02 Cybios Llc Pluripotente embryonlignende stamceller, præparater, fremgangsmåder og anvendelse deraf
WO2005113754A1 (fr) * 2004-05-20 2005-12-01 New York Medical College Cellules souches adultes multipotentes
WO2006088867A2 (fr) * 2005-02-15 2006-08-24 Medistem Laboratories, Incorporated Procede pour l'expansion de cellules souches
EP1815741B1 (fr) * 2006-01-19 2011-07-06 Medestea Research & Production S.p.A. L'utilisation de compositions contenants des dérivés des pipéridine pour protéger des systèmes biologiques
US8241621B2 (en) * 2006-12-18 2012-08-14 Medistem Laboratories Stem cell mediated treg activation/expansion for therapeutic immune modulation

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105018428A (zh) * 2015-05-27 2015-11-04 贵州北科泛特尔生物科技有限公司 脐带血造血干细胞的体外扩增方法
CN105670987A (zh) * 2016-03-21 2016-06-15 杭州市萧山区中医院 一种毛囊干细胞诱导分化为血管内皮细胞的抑制方法
CN105670987B (zh) * 2016-03-21 2019-04-02 杭州市萧山区中医院 一种毛囊干细胞诱导分化为血管内皮细胞的抑制方法
KR20190026772A (ko) * 2016-07-01 2019-03-13 고쿠리츠다이가쿠호진 도호쿠다이가쿠 장기 섬유증의 예방 또는 치료제
EP3479833A4 (fr) * 2016-07-01 2020-02-26 Tohoku University Agent de traitement prophylactique ou thérapeutique pour la fibrose des organes
AU2017287529B2 (en) * 2016-07-01 2022-11-10 Life Science Institute, Inc. Prophylactic or therapeutic agent for organ fibrosis
KR102513507B1 (ko) * 2016-07-01 2023-03-22 고쿠리츠다이가쿠호진 도호쿠다이가쿠 장기 섬유증의 예방 또는 치료제
CN108441474A (zh) * 2018-03-23 2018-08-24 天晴干细胞股份有限公司 一种来源于脐带血、胎盘造血干细胞分离纯化的方法
CN108441474B (zh) * 2018-03-23 2021-10-08 天晴干细胞股份有限公司 一种来源于脐带血、胎盘造血干细胞分离纯化的方法
CN109364090A (zh) * 2018-12-06 2019-02-22 西华大学 油橄榄叶提取物在制备防治胎儿酒精综合症药物中的应用
CN109364090B (zh) * 2018-12-06 2020-10-23 西华大学 油橄榄叶提取物在制备防治胎儿酒精综合症药物中的应用
WO2020146874A1 (fr) * 2019-01-11 2020-07-16 Figene, Llc Cellules fibroblastes régénératrices

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