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WO2008070868A1 - Sécrétions de cellules souches et procédés associés - Google Patents

Sécrétions de cellules souches et procédés associés Download PDF

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Publication number
WO2008070868A1
WO2008070868A1 PCT/US2007/086878 US2007086878W WO2008070868A1 WO 2008070868 A1 WO2008070868 A1 WO 2008070868A1 US 2007086878 W US2007086878 W US 2007086878W WO 2008070868 A1 WO2008070868 A1 WO 2008070868A1
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Prior art keywords
stem cell
cells
product
cell secretions
secretions
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Keith Skinner
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American Symbolic LLC
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American Symbolic LLC
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/98—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution of animal origin
    • A61K8/981—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution of animal origin of mammals or bird
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin

Definitions

  • the present disclosure relates to the field of stem cell conditioned media and more particularly to stem cell secretions, lyophilization, and stabilization of the secretions.
  • the constituents of the stem cell secretion may be protected with fatty acids to both prevent premature breakdown and improve cellular uptake when used as an epidermal stem cell secretion composition for topical and systemic administration, the compounding of the stem cell secretion composition to provide cosmetic formulations suitable for therapeutic use and test methods for determining the bio-active effectiveness of the stem cell secretion.
  • Stem cell secretions are derived from epithelial stem cell conditioned media.
  • the stem cell secretions are then applied topically, orally, or rectally, etc., to derive health benefits from the growth factors and other molecules comprising the stem cell secretion.
  • the stem cell secretion may optionally be modified by covalently bonding fatty acids to protect the molecules through the delivery process and to make them more readily available to cells.
  • a method comprising harvesting stem cells, cultuhng the stem cells, separating the a stem cell conditioned media from the stem cells and other debris to produce purified stem cell secretions, and providing the stem cell secretions to be delivered to an animal.
  • a method is comprising harvesting stem cells, culturing the stem cells, removing a stem cell conditioned media having stem cell secretions, adding a fatty acid chloride source to the aqueous solution at pH 1 1-14, stirring, purifying the resulting precipitate comprising at least fatty acid modified stem cell secretions.
  • a composition comprising stem cell secretions harvested from a conditioned stem cell media and separated from the stem cells from which the stem cell secretions are derived, wherein the stem cell secretions have at least one fatty acid chemically attached thereto.
  • Fig. 1 is a flow diagram of an embodiment of a method for creating a stem cell secretions additive
  • Fig. 2 is a flow diagram illustrating an embodiment of a process for creating stem cell secretions
  • Fig. 3 is a flow diagram illustrating an embodiment of a process for modifying stem cell secretions with fatty acids.
  • stem cell additive or “stem cell secretion(s)” shall be understood to mean secretions of stem cells or non- stem cells grown in the same media with the stem cells.
  • the secretions expressly include growth factors, regulatory proteins, hormones, signaling proteins, or other organic molecules secreted from the stem cells or non-stem cells grown in the same media as the stem cells.
  • Stem cells are primal undifferentiated cells, present in small numbers among differentiated cells in tissues and organs. Stem cells have the ability to differentiate into different cell types to act as a mechanism of repair for damaged tissues. They secrete various signaling factors such as proteins that have the ability to maintain and renew aging tissues.
  • stem cells There are two types of stem cells: those found in adult tissues (“Somatic Stem Cells”), which are “multipotent,” because they multiply themselves into cells of a specific type or family of cells; and embryonic stem cells, which are able to give rise to all the cell types seen in an adult organism, and are therefore said to be “totipotent.”
  • Somatic Stem Cells those found in adult tissues
  • embryonic stem cells which are able to give rise to all the cell types seen in an adult organism, and are therefore said to be “totipotent.”
  • specialized cells cannot backtrack and start over. Generally, the most cells are able do is to return from a non-dividing state to a dividing one, as for example connective tissue and bone cells do in healing a wound or break, and as skin cells do in regenerating skin lost through burns or cuts. It is believed that nerve cells can regrow their axons and dendrites, but nerve cells themselves cannot be replaced.
  • adult cell types can dedifferentiate slightly, for example, as some connective tissue cells do to produce new bone cells. Generally, more extensive differentiation is considered abnormal and is a diagnostic sign of certain cancers.
  • Adult hair follicles provide an example of normal healthy crossover between different organ adult stem cells, as they can occasionally act as bone or fat stem cells, and may differentiate into bone or fat.
  • stem cells that demonstrate a high degree of plasticity.
  • researchers are investigating adult stem cells from a variety of sources, including, skin/hair follicles, adipose (fatty) tissue, the synovial membrane (inner layer of lubricating capsule surrounding free moving joints), perivascular cells (related to or occurring in tissues surrounding blood vessels), and tooth dental pulp cells.
  • the adult stem cells isolated from the skin (“dermis”) were shown to be able to differentiate in laboratory culture dishes giving rise to muscle and neurons.
  • the observable characteristics produced by the interaction of genetic make up and environmental factors, as well as the origin of this stem cell population is unclear, as the initial dermal population was heterogeneous (no uniform composition).
  • Hair follicle epithelial cells have been the subject of intense study in relation to skin renewal and tumor biology. As seen during the course of normal follicle growth and cycling, dynamic epithelial (membrane linings of cavities or tubes)-mesenchymal (loosely organized undifferentiated mesodermal, or middle layer, cells that give rise to such structures as connective tissues, blood, lymphatics, bone, and cartilage) persists from embryonic development into adulthood.
  • Hair follicles contain discrete populations of interacting cells that are clustered in defined sites and can be isolated, cultured, and then experimentally manipulated.
  • the hair follicle is emerging as a major developmental stem- cell model, encompassing paradigms of epithelial-mesenchymal interactions and epithelial stem cell behavior, as well as high accessibility in the adult body.
  • the follicle dermis (skin) acts as an important stem cell repository for repair of the dermis after skin wounding.
  • the stem cell hematopoietic (blood stream) activity was demonstrated in the follicle dermis.
  • the skin is currently one of the few organs in which adult stem cells can be maintained and propagated in the laboratory.
  • stem cells With respect to the epidermis, cells are generated through proliferation that occurs only in the basal layer; therefore, stem cells must be located there.
  • keratinocytes display such heterogeneous proliferative characteristics.
  • the main source of the stem cells responsible for continual epidermal renewal appears to reside in the center of the EPU (epidermal proliferative unit). Similar to this concept, the hair follicle generates a terminally differentiated keratinized end product, the hair shaft that is eventually shed.
  • Keratinocytes within the budge area may depend on their environment or niche for maintaining their stem cell characteristics.
  • the bulge area also houses melanocyte stem cells, which are normally quiescent but proliferate at anagen onset to repopulate the new lower anagen hair follicle with melanocytes that generate melanin leading to pigmentation of their hair.
  • epidermal stem cells and hair follicle stem cells constitute two distinct populations and understanding of differences in their structures and means of communication with other cells is of importance for developing treatments based on stem cells.
  • the development of the hair follicles represents a series of interactions between cells derived from embryonic ectoderm and mesoderm. Both an increase and a decrease in the expression of various growth factors, adhesion molecules, glycosaminoglycans, etc., has been seen during this development process. Yet, their roles in different stages of hair cycle have not been identified. It is generally assumed that follicular development and hair cycling is a consequence of messages passing between the epidermal compartment of the follicle and the dermal compartment. The epidermal compartment was shown in animal experiments to play a role in follicle development, the fate of the emerging hair structure, and provide signals for a new round of hair growth or maintenance of the anagen phase.
  • hDP human scalp-derived dermal papilla
  • human DP cells failed to proliferate out of intact papilla when maintained in a growth medium specifically designed for human keratinocyte growth, (keratinocyte growth medium KGM).
  • Human scalp-derived papilla (hDP) cells grew when plated together with human epithelial keratinocytes (hK), which suggests that hK support growth either directly (transmission of contact-dependent growth signals) or indirectly (transmission of diffusible growth signals).
  • the skin healant properties of the conditioned medium of cultured human keratinocytes (KCM) containing the autocrine and paracrine activities of growth factors they produce was shown by the ability of cultured human keratinocytes (KCM) to display growth stimulating properties on several cell lines involved in wound healing process as well as the ability of KCM to stimulate re- epithalization of wounds in human skin.
  • Collagenases the enzymes that degrade collagen, regulate such process in wound healing as the migration of keratinocytes over the wound bed, angiogenesis, and remodeling of the granulation tissue. Degradation of collagen is necessary for the wound healing process.
  • cultured human keratinocytes and fibroblasts were stimulated with KCM and assessed for collagenase production by binding studies with tritiated diisopropylfluorophosphate (3H-DFP) and zymography. Lysates from unstimaluted human keratinocytes contained 3H-DFP binding proteins of both 72 and 92 kDa.
  • Conditioned media is a soup produced by the secretory output of growing epithelial stem cells in culture; this soup is known to facilitate normal growth of skin and hair follicle cell types.
  • HGF Hepatocyte Growth Factor
  • the extracellular matrix is made of a wide variety of components with predominance of collagens and noncollagenous proteins. These two groups of compounds contribute to the maintenance of tissue integrity and architecture. In addition, they affect cell behavior. Dermatopontin is one of the noncollagenous components of the ECM. In humans, dermatopontin is detected in fibroblasts, in the heart, muscle, and lungs. In skin, dermatopontin tends to be distributed around collagen fibers and within endothelial cells.
  • Dermatopontin plays a vital role in ECM architecture (interaction with decorin and modification of collagen fibrillogenesis), cell behavior (a weak adhesion activity for certain fibroblasts and neurogenic cells; interaction with TGF-beta bioactivity and modification of collagen fibrillogenesis, possibly by interacting with collagen molecules), and pathological involvements (increased expression around myocardial infarct zone, decreased expression in leimyoma and keloid, and decreased expression in fibrosing diseases).
  • a change in the dermatopontin expression also has been reported in the chondrocyte culture system.
  • scientistss suggest that dermatopontin could interact with type I collagen and modify its fibrillogenesis thus aiding at maintaining the mechanical strength or elasticity of the cartilage.
  • fibroblasts are capable of producing stem cell factors (SCF) and mast cell differentiation factors.
  • SCF stem cell factors
  • mast cell differentiation factors are a multifactorial regulator of hematopoietic stem cell, mast cell, and melanocyte differentiation and function.
  • keratinocytes are capable of producing SCF
  • the inventors investigated whether keratinocytes can produce mast cell differentiation. The results have shown that, using human HaCaT keratinocyte cell lines, the keratinocyte supernatants produce and release factors that upregulate mast cell characteristics. It was shown that differentiating keratinocytes displayed much higher ability to release these factors than proliferating HaCaT keratinocytes. This activity is not likely to be due to SCF since human mast cell line HMC-1 cells have been shown to be poor responders to SCF. The inventors concluded that keratinocytes release both SCF and differentiation-dependent factors which regulate mast cell development.
  • Keratinocyte-induced dendricity of cultured melanocytes is triggered by the early secretion of at least one unidentified factor(s) by keratinocytes. Approximately one-half of factors showing growth promoting activity from conditioned medium, as well as most of the dendricity and melanization stimulating activities were of low (less than 500 Daltons) molecular weight.
  • the culture of hair cell follicle derived stem cells has been performed by the methods disclosed in U.S. Patent No. 5,556,783, which is incorporated by reference herein.
  • the stimulation of their growth has been performed by several different methods, including the teachings of U.S. Patent No. 5,902,741 , which is likewise incorporated by reference, who utilize a three-dimensional culture medium and TGF-beta.
  • U.S. Patent Nos. 5,962,325, and 6,022,743, which are incorporated by reference teaches the use of a similar methods for the culture of mesenchymal cells and pancreatic parenchymal cells.
  • Extracellular collagen matrices as disclosed in U.S. Patent Application No. 2005/0106723, which is incorporated by reference, have also been used.
  • Other inventors have isolated a specific polypeptide from skin cell culture that stimulates epithelial cell growth in U.S. Patent Application No. 2003/0040471 , which is also incorporated by reference.
  • the inventors have discovered that the stimulation of a stem cell culture, specifically cells derived from the bulge area of a hair follicle, results in substantial increase in both the expansion of the cell population and its release of signaling factors, growth factors, and other biological molecules into the conditioned growth media. Moreover, the inventors have discovered that the stem cell secretions derived according to the instant teachings may be modified by bonding fatty acids to their active sites, thereby protecting the stem cell secretions and creating a vehicle for more efficient delivery intracellular ⁇ .
  • stem cell secretions are harvested in operation 100, which is shown in greater detail in Fig. 2.
  • the stem cell secretions may then be optionally modified with fatty acids in operation 200, which is illustrated in greater detail in Fig. 3.
  • the resulting stem cell secretions or fatty acid modified stem cell secretions may be used, for example, as additives in a variety of products, from lotions and skin creams to orally consumed therapeutic products to cooking oils in operation 300.
  • one or more hair follicles comprising at least one actively growing epidermal stem cell are extracted and the at least one hair follicle is enzymatically treated to separate them from the one or more hair follicles in operation 1 10.
  • the one or more epidermal stem cells are cultured to provide a first population of stem cells and the first population of stem cells is purified by methods well understood in the art.
  • the first population of stem cells is then cultured to provide a second population of stem cells, as is well understood in the art.
  • the second population of stem cells may be transferred to a second medium to culture the second population of stem cells.
  • the medium may comprise Eagle's serum free alpha-MEM, 10-8 M dexamethasone, 1OmM sodium beta- glycerophosphate, and 50 mg/ml L-ascorbic acid 2-phosphate.
  • the second population of stem cells may be stimulated with at least one low intensity pulsed ultrasound source to provide a third population of stem cells and a conditioned medium having stem cell secretions (the first and second mediums also have stem cell secretions that can be harvested).
  • the third population of stem cells may be separated from the conditioned medium in operation 130, and the conditioned medium may be filtered to remove cells and debris in operation 140.
  • the conditioned medium may be filtered to sterilize the medium, and the stem cell secretions may be freeze-dried and lyophilized to provide one or more epidermal stem cell additives in operation 150.
  • the conditioned media having stem cell secretions made according to the above disclosed method comprises a "conditioned soup" produced by the secretory output of growing skin stem cells in a culture medium.
  • the skin stem cells may comprise material obtained from a bulge location of one or more hair follicles.
  • the resulting "conditioned soup" may comprise a variety of factors which may facilitate therapeutic treatment of the epidermal structure of a skin tissue. Such factors include, but are not limited to: hepatocyte growth factor (HGF), dermatopontin, and other stem cell growth factors and the like.
  • HGF hepatocyte growth factor
  • the epidermal stem cell additive may comprise a powder form, although it will be appreciated that the additive may be formulated in any suitable manner as is understood in the art.
  • the first population of stem cells may be obtained in a yield of about 10 to about 2000 cells during culturing. Furthermore, in another aspect the first population of stem cells may be obtained in a yield of about 50 to about 1000 cells. In yet another embodiment the first population of stem cells may be obtained in a yield of about 100 to about 200 cells.
  • these cells may be obtained in a yield of about 25,000 to about 2,500,000 cells.
  • the second population of stem cells may have a yield of about 125,000 to about 1 ,250,000 cells.
  • the second population of stem cells may have a yield of about 250,000 cells.
  • the third population of stem cells may be expanded to a range between about 400,000 and about 40,000,000 stem cells. Furthermore, in another aspect the third population of stem cells may be expanded to a range between about 800,000 to about 20,000,000 stem cells. Alternatively, in yet another embodiment the third population of stem cells may expanded to about 4,000,000 stem cells during culturing.
  • the low intensity pulsed ultrasound source may have a signal speed ranging from about 0.15 MHz to about 15 MHz, an intensity of about 7 mW/cm 2 to about 700 mW/cm 2 and the source may be applied for about 2 minutes to about 400 minutes during each 24 hour interval of an about 24 hour to about 720 hour time period.
  • the low intensity pulsed ultrasound source may have a signal speed ranging from about 0.30 MHz to about 7.5 MHz, an intensity of about 14 mW/cm 2 to about 350 mW/cr ⁇ 2 and the source may be applied for about 4 minutes to about 200 minutes per 24 hours of an about 24 hour to about 360 hour time period.
  • the low intensity pulsed ultrasound source may have a signal speed of about 1.5 MHz, an intensity of about 70mW7cm 2 , and the source may be applied for about 20 minutes to about 40 minutes during each 24 hour interval of a time period of about 72 hours.
  • the constituents of the resulting stem cell secretions may be modified by complexing with fatty acids as described herein.
  • the stem cell secretions may be filtered to remove cells and debris and may comprise a filter having a pore size of about 2 microns to about 10 microns. In another aspect the filter may have a pore size of about 5 microns.
  • a filter having a pore size of about 2 microns to about 0.02 microns may be used.
  • the sterilizing filter may be about 0.2 microns in pore size.
  • the stem cell secretions may be stabilized with at least one of ethylenediaminetetraacetic acid (EDTA) and ethylene glycol bis(2-aminoethylether)-N,N,N'N'-tetraacetic acid (EGTA) or any other stabilizer as is known in the art.
  • EDTA ethylenediaminetetraacetic acid
  • EGTA ethylene glycol bis(2-aminoethylether)-N,N,N'N'-tetraacetic acid
  • the present disclosure also discloses a method for modifying the constituents of the stem cell secretions with lipids thereby making the constituents of the stem cell secretions more readily bioavailable.
  • adding fatty acids to constituents of the stem cell secretions further allows the skin to more easily and efficiently absorb the constituents of the stem cell secretions.
  • adding fatty acids to constituents of the stem cell secretions creates both a vehicle for delivery through lipid bilayers of cells and the skin, and a "time release" effect as the constituents of the stem cell secretions are not bioavailable until the lipid side chains of the modified constituents of the stem cell secretions are cleaved.
  • Fatty acids are enzymatically cleaved carbon by carbon.
  • the present disclosure proposes a novel method of making constituents of the stem cell secretions more deliverable to cells by adding fatty acids to active sites on the constituents of the stem cell stem cell secretions.
  • the fatty acids are covalently bonded to one or more active sites of the constituents of the stem cell secretions, thereby preventing the constituents of the stem cell secretions from reducing free radicals prior to delivery of the constituents of the stem cell secretions at the target location.
  • Constituents of the stem cell secretions will have at least one active site that can reversibly react with the carboxyl end of fatty acids; these may include NH 2 , SH 2 , and OH sites, and others as will be known and understood by artisans. Indeed, these sites are preferentially bound in the following order: amino or any free binding site, then sulphydryl or any free binding site, and finally hydroxyl sites. As will readily be recognized by artisans, NH 2 sites are will be modified first due their positive charge.
  • modification of the hydroxyl active sites is advantageous because ether bonds form between the fatty acid and the constituents of the stem cell secretions.
  • the ether bonds are more stable in biologic systems, which means that the cell takes longer to break down the fatty acid and expose the active site.
  • constituents of the stem cell secretions that may be modified according to the present disclosure include, but are not limited to, constituents of the stem cell cultured media such as proteins, antioxidants (for example glutathione, hyaluronic acid, carnosine, and others), and other molecules having at least one active site that can be modified by covalent bonding of a fatty acid.
  • constituents of the stem cell cultured media such as proteins, antioxidants (for example glutathione, hyaluronic acid, carnosine, and others), and other molecules having at least one active site that can be modified by covalent bonding of a fatty acid.
  • fatty acids comprise an aliphatic chain coupled to a carboxylic acid.
  • the carboxy end of fatty acids are reacted to the active sites of the constituents of the stem cell secretions.
  • the fatty acid-stem cell secretions constituents complex serves two purposes. First, the fatty acids reversibly block the active sites of the constituents of the stem cell secretions until the constituents of the stem cell secretions are delivered intracellular ⁇ . Second, the lipophilic aliphatic side chain or chains of the fatty acids allow the constituents of the stem cell secretions to more readily cross the cell membrane and penetrate skin, for example.
  • any fatty acid having 2 or more carbons in the aliphatic chain are suitable to be coupled to constituents of the stem cell secretions.
  • the fatty acids may be saturated or unsaturated.
  • Fatty acids having more than 35 carbons and fatty acids having aliphatic chains of both an even and an odd number of carbons are equally applicable with the teachings of the present disclosure.
  • unsaturated fatty acids having any number of double or triple bonds in both -cis or -trans configurations are expressly contemplated.
  • myristoleic acid C14:1
  • palmitoleic acid C16:1
  • oleic acid C18:1
  • linoleic acid C18:2
  • ⁇ -linoleic acid C18:3
  • arachidonic acid C20:4
  • eicosapentaenoic acid C20:4
  • eicosapentaenoic acid C20:5
  • erucic Acid C22:1
  • docosahexaenoic acid C22:6 are examples of common unsaturated fatty acids that may be coupled to constituents of the stem cell secretions according to the present disclosure.
  • the fatty acids of the present disclosure may be oils, such as olive oil, jojoba oil, sunflower oil, safflower oil, rapeseed oil, corn oil, soya oil, wheat germ oil, cottonseed oil, almond oil or oils of other nuts, palm oil, coconut oil, vegetable oil, butter, lard, as well as other oils comprising, at least in part, fatty acids.
  • oils such as olive oil, jojoba oil, sunflower oil, safflower oil, rapeseed oil, corn oil, soya oil, wheat germ oil, cottonseed oil, almond oil or oils of other nuts, palm oil, coconut oil, vegetable oil, butter, lard, as well as other oils comprising, at least in part, fatty acids.
  • the oil or fatty acid must be non-toxic.
  • the oil selected my comprise oils known to be nutritionally healthy, such as olive oil or omega-3 fatty acids. Use of such health-type oils may be of interest to the health food markets, etc.
  • the stem cell secretions may comprise or have added it to health food supplements, the total product of which may then to be sold as such or may be included with other additives, such as antioxidants, zinc oxides, or titanium dioxides, in skin creams or other cosmetic applications, for example.
  • Other examples include cooking or dipping oils for oral consumption having the fatty acid modified stem cell secretions.
  • the process for protecting constituents of the stem cell secretions with fatty acids is performed in an aqueous solution using the fatty acid chloride of the fatty acids being used to modify the active sites of the constituents of the stem cell secretions in operation 210.
  • the concentration of the stem cell secretions dissolved into aqueous solution may be as high as possible, according to embodiments. According to other embodiments, a more dilute concentration may be used to reduce steric hindrance for complete fatty acid coupling to large constituents.
  • the pH is raised to pH 1 1-14 with a base in operation 220.
  • the base is an inorganic base, such as NaOH, which prevents undesirable side reactions.
  • the pH is kept in the range of pH 1 1-14 to drive the modification reaction.
  • the fatty acid chloride is added to drop-wise to the solution in operation 230 under agitation/stirring in operation 240, together with additional base to maintain the desired pH.
  • the fatty acid chloride is added to the active site(s) of each constituent of the stem cell secretions, the resulting product falls out of solution as a precipitate.
  • the solution need not have the pH raised before adding the fatty acid chloride and the base, whereby the pH will be raised as a matter of course during the reaction.
  • the precipitate comprising the fatty acid modified stem cell secretions
  • Harvesting may occur simply by decanting the water, washing the precipitate with water at least once, and drying.
  • the resultant dry precipitate comprises the constituents of the stem cell secretions coupled to one or more fatty acid molecules.
  • the precipitate may then be added as an additive to other products such as vitamin tablets, lotion, skin creams, etc., for delivery purposes as described subsequently. According to embodiments, nearly any product having the fatty acid modified stem cell secretions by the disclosed process are expressly contemplated.
  • the methods of the instant disclosure may be performed on a large scale without appreciable changes to the principles disclosed by the exemplary protocol.
  • the fatty acid modified stem cell secretions constituent complex may be further modified, either before or after the process disclosed herein to provide further desirable characteristics.
  • antioxidant molecules such as glutathione
  • glutathione may be esterified prior to the process disclosed herein.
  • Other similar modifications that are known in the art, such as acetylation with glutathione, are also possible and expressly contemplated, provided active sites on the constituents of the stem cell secretions are available for modification.
  • EXAMPLE 1 One gram of stem cell secretions (conditioned media) from CK-15/CK-
  • EXAMPLE 1 the procedure of EXAMPLE 1 is duplicated. However, rather than using palmitic acid as the fatty acid, olive or jojoba oil chlorides are added as the fatty acid chloride. Artisans will readily recognize and understand the process of making the olive or jojoba oil chloride.
  • EXAMPLE 3 Similarly, the procedure of EXAMPLE 1 is duplicated. However, rather than using palmitic acid as the fatty acid, one or more of the other fatty acids listed above is added as the fatty acid chloride. Artisans will readily recognize and understand the process of making the desired fatty acid chloride.
  • Oils that have multiple fatty acids, each having different sized aliphatic chains may be used to create "time-release" stem cell secretion preparations. Shorter aliphatic chains are cleaved more quickly to expose the active site of the constituents of the stem cell secretion preparations, while the longer aliphatic chains are protected for longer. Thus, the net effect is an extended delivery time for the modified stem cell secretion preparations. Additionally, other growth factors, antioxidants, coenzymes, and biologically desirable or necessary molecules may be added to the stem cell secretions prior to adding fatty acids as supplements. EXAMPLE 5
  • stem cell secretions include at least one of senescence, dermatopontin, ICAM1 , IGF-3, Insulin-like growth factor binding protein 7, pleitrophin, vimentin, and ld-1 gene.
  • Senescence induces a decrease of dermatopontin, ICAM-1 , collagen 1 and 3, insulin-like growth factor binding protein 3, pleitrophin, HSP-27, SOD 1 , and vimentin, and an increase of collagen 8, MMP-1 and MMP-3, heme oxygenase-1 , insulin-like growth factor binding protein 7, and PGD2 synthase.
  • Dermatopontin is an extracellular matrix protein with possible functions in cell-matrix interactions and matrix assembly.
  • ICAM1 (CD54) is typically expressed on endothelial cells and cells of the immune system. ICAM1 binds to integrins of type CD1 Ia / CD18, or CD1 Ib / CD18. ICAM1 is also exploited by Rhinovirus as a receptor. Insulin- like growth factor binding protein 3. The protein forms a ternary complex with insulin-like growth factor acid-labile subunit (IGFALS) and either insulin-like growth factor (IGF) I or II.
  • IGFALS insulin-like growth factor acid-labile subunit
  • Insulin-like growth factor binding protein 7 this protein binds insulin altering its interaction with cell surface receptors.
  • Pleitrophin Consistent with its role in promoting keratinocyte growth, PTN was upregulated during cutaneous wound healing in vivo.
  • Vimentin Cytoskeleton protein involved in cell shape changes during senescence, ld-1 gene. Generally, proliferating cells express multiple Id genes, whereas upon differentiation in many cell types, the expression of Id genes is down- regulated. The expressions of Id1, 1d2, and 1d3 are induced when the serum or growth factors are added to Go-arrested fibroblasts. In addition, abolishing Id proteins synthesis by antisense oligonucleotides blocks the quiescent fibroblasts into the cell cycle.
  • higher intensity ultrasound causes the secretion of healing factors from the stem cells into the stem cell secretion.
  • Low- intensity pulsed ultrasound (LIPUS) was used for its distinct effects on biologic mineralization at intensities of ⁇ 100 mW/cm2. Intensity-dependent differences in the pattern of accelerated mineralization may be due to different alterations in regulation of collagenous matrix formation.
  • LIPUS low- intensity pulsed ultrasound
  • a pharmaceutically acceptable cosmetic base may be enriched while adding the fatty acid-stem cell secretion additive and one or more stabilizers using one or more high frequency ultrasonification methods to provide one or more enriched cosmetic bases.
  • a pharmaceutically acceptable cosmetic base may be enriched while adding the fatty acid-stem cell secretion additive and one or more stabilizers using one or more high frequency ultrasonification methods to provide one or more enriched cosmetic bases.
  • the enriched cosmetic base of may further comprise one or more modified glutathiones which may have important therapeutically additive properties as is known in the art.
  • the enriched cosmetic base may comprise one or more modified carnosines which also may have important therapeutically additive properties as is known in the art.
  • the enriched cosmetic base may further comprise one or more modified glutathiones and one or more modified carnosines and the combination of the enriched cosmetic base, the one or more glutathiones and the one or more carnosines may be further lyophilized to dryness for future use.
  • further stabilizers and ingredients suitable for compounding therapeutically active formulations may also be added.
  • the therapeutic efficacy of any of the enriched cosmetic bases described above may be tested for skin penetration by applying any of the enriched cosmetic bases to a portion of a skin tissue and observing the amount of penetration of the enriched cosmetic base into the skin tissue as is understood in the art. Furthermore, in another embodiment the therapeutic efficacy of any of the enriched cosmetic bases described above may be tested for bioavailability by applying the enriched cosmetic base to a portion of a skin tissue and observing the bioavailability of the enriched cosmetic base within the skin tissue.
  • one or more compositions comprising one or more stem cell secretions as described above may further comprise a pharmaceutically acceptable carrier to make the composition effective for systemic administration.
  • the efficacy of this composition may be tested by applying the enriched cosmetic base composition to a portion of a skin tissue and observing the bioavailability of the composition within the skin tissue.
  • a dosage form of one or more stem cell secretions, both fatty acid modified and unmodified may further comprise one or more pharmaceutically acceptable carriers.
  • the dosage form may be compounded and comprise one or more of the following: a tablet, a capsule, injection, a liquid, a powder, a lecithin granule, a liposome, an inhalant, a sublingual form, a suppository, an oral spray, dermal patches, creams, gels, lotions, masks, other topical applications, and the like as is understood in the art.
  • a tablet a capsule
  • injection a liquid
  • a powder a lecithin granule, a liposome
  • an inhalant a sublingual form
  • a suppository an oral spray, dermal patches, creams, gels, lotions, masks, other topical applications, and the like as is understood in the art.
  • fatty acid modified stem cell secretion taken orally are protected in the digestive tract and more readily absorbed to a greater extent than unprotected molecules.
  • one or more enriched cosmetic bases may comprise one or more of the following: cocoa butter, aloe vera gel, aquafor, petroleum jelly, lecithin, almond oil, borage oil, canola oil, grape seed oil, jojoba oil, olive oil, soybean oil, sunflower oil, wheat germ oil, apricot kernel oil, carrot oil, coconut oil, hemp seed oil, flax seed oil, mango butter, evening primrose oil, black currant oil, avocado oil, microcrystalline wax, paraffin, petrolatum, petroleum jelly, ozokerite, montan wax, beeswax, at least one of lanolin and a derivative of lanolin, candelilla wax, ouricury wax, carnauba wax, Japan wax, cocoa butter, sugarcane wax, cork fiber wax, and the like as is understood in the art
  • the enriched compositions may be compounded to be systemically effective.
  • compositions described herein may comprise one or more pharmaceutically acceptable carriers comprising one or more of the following: a binding agent, a filler, a lubricant, a disintegrant, a wetting agent, a sugar, a starch, a cellulose and derivatives thereof, a stabilizer, a tableting agent, an antioxidant, a preservative, a colorant, a flavorant, and the like as is known in the art.
  • enriched cosmetic base may be effective in rejuvenating a portion of an epithelial structure of a human skin, according to embodiments.
  • one or more of these topically active dosage forms may be applied topically for a period of from at least one to at least three times daily to a portion of an epithelial structure of a human skin.
  • one or more topically active dosage forms as described above may be effective in restoring hair growth of a portion of an epithelial structure comprising one or more hair follicles.
  • one or more topically active dosage forms may be applied to a portion of one or more epithelial structures of skin tissues and may be effective in reducing wrinkles, reducing cross-linking, controlling free radical deterioration, increasing collagen levels, increasing growth factors, reducing lipofuscin, reducing one or more inflammatory conditions, reducing benign epidermal proliferation or cancers, rejuvenating one or more traumatic conditions, and controlling dermal remodeling and scar formation to regrow skin tissue without abnormal appearance.
  • one or more enriched cosmetic bases compounded as topically active dosage forms may further comprise alpha-hydroxy lactic acid.
  • these topically active dosage forms may be effective in reducing cohesive dermatomes after application to a portion of one or more skin tissues.
  • either one or more orally active or topically active dosage forms may protect against age-related decline, promote good health and slow premature aging by administering one or more enriched cosmetic bases (alone or in combination as a compounded composition) to one or more mammals, such as humans.
  • these one or more orally active or topically active dosage may be administered according to one or more schedules as shown in the following: a single daily dose, in divided daily doses, in doses every other day, or in doses every three days and the like so as to insure proper effectiveness of the therapy, according to various embodiments.
  • one or more orally active or topically active dosage forms may comprise one or more of the following: a tablet, a capsule, an injection, a liquid, a powder, a lecithin granule, a liposome, an inhalant, a sublingual tablet, a suppository, an oral spray, a dermal patch, creams, gels, lotions, masks, other topical applications, and the like as is understood in the art.
  • one or more orally active or topically active dosage forms may be further configured with one or more pharmaceutically acceptable carriers of the following dosage forms: a tablet, a capsule, an injection, a liquid, a powder, a lecithin granule, a liposome, an inhalant, a sublingual tablet, a suppository, an oral spray, a dermal patch, creams, gels, lotions, masks, other topical applications, and the like as is understood in the art.
  • These may contain fatty acid modified or unmodified stem cell secretions, or fatty acid modified or unmodified stem cell secretions and other supplements as described previously.
  • one or more body organs of a mammal may be rejuvenated by systemically administering one or more epidermal stem cell additives or compositions as described above.
  • the one or more body organs may be the mammal's heart or kidney and the like, as is understood in the art and one or more compositions as described above may be to a mammal for the treatment of hepatic failure or to promote healing of fractures of a bone.
  • other benefits of administering one or more epidermal stem cell additives and compounds thereof may be to promote longevity in a mammal, quell inflammation in a mammal or reduce the risk of degenerative diseases in a mammal.
  • a cosmetic base in a method of testing the therapeutic effectiveness of an epidermal stem cell additive a cosmetic base is provided; a composition comprising one or more stem cell additives is provided (as described herein); the cosmetic base and the epidermal stem cell additive are compounded to provide a cosmetic foundation; the compounded cosmetic foundation is applied to a portion of a surface of a skin tissue; the amount of penetration of the cosmetic foundation into the skin tissue is observed; and the bioavailability of the cosmetic foundation within the skin tissue is determined.
  • at least a portion of the at least one stem cell additive is bound to at least one bio-marker configured to identify bioavailability of the cosmetic foundation within the skin tissue.
  • the marker may comprise magnetite in the form of Minden beads and the like as is understood in the art.
  • the at least one stem cell secretion additive may be attached to at least one bio-marker, non- binding cells may be rinsed away from the at least one stem cell additive to leave a bound substantially bio-active residue; and the bound substantially bio-active residue may be enzymatically treated (as is known in the art) to provide an unbound substantially bioactive residue.
  • each step according to this method may be repeated upon the bioactive residue until the purity of the at least one stem cell additive has reached a predetermined suitable level.

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  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Zoology (AREA)
  • Birds (AREA)
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  • Dermatology (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention concerne des sécrétions de cellules souches dérivées d'un milieu conditionné de cellules épithéliales. Les sécrétions de cellules souches sont ensuite appliquées par voie topique, orale ou rectale, etc., afin de tirer les bénéfices pour la santé des facteurs de croissance et des autres molécules contenues dans les sécrétions de cellules souches. Les sécrétions de cellules souches peuvent éventuellement être modifiées par liaison covalente avec des acides gras afin de protéger les molécules lors du procédé de délivrance et de les rendre plus facilement disponibles pour les cellules.
PCT/US2007/086878 2006-12-07 2007-12-07 Sécrétions de cellules souches et procédés associés Ceased WO2008070868A1 (fr)

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
US86897106P 2006-12-07 2006-12-07
US60/868,971 2006-12-07
US95401407P 2007-06-19 2007-06-19
US60/954,014 2007-06-19
US95253507P 2007-07-27 2007-07-27
US60/952,535 2007-07-27

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WO2008070868A1 true WO2008070868A1 (fr) 2008-06-12

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Publication number Priority date Publication date Assignee Title
WO2015001124A1 (fr) 2013-07-05 2015-01-08 Université Catholique de Louvain Milieu conditionné provenant de cellules souches de foie humain adulte et utilisation de ce milieu dans le traitement de maladies
US20200157500A1 (en) * 2017-06-30 2020-05-21 Cha Biotech Co., Ltd. Stem cell-derived skin precursor cell culture medium and preparation method therefor
US10744160B2 (en) 2014-12-01 2020-08-18 T-Helper Cell Technologies, Llc Stem cell material and method of manufacturing
US11376283B2 (en) 2016-05-31 2022-07-05 T-Helper Cell Technologies, Llc Stem cell material, compositions, and methods of use

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US6620419B1 (en) * 1998-09-15 2003-09-16 Sederma Cosmetic or dermopharmaceutical use of peptides for healing, hydrating and improving skin appearance during natural or induced ageing (heliodermia, pollution)
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US7118746B1 (en) * 1999-05-14 2006-10-10 Skinmedica, Inc. Conditioned cell culture medium compositions and methods of use

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015001124A1 (fr) 2013-07-05 2015-01-08 Université Catholique de Louvain Milieu conditionné provenant de cellules souches de foie humain adulte et utilisation de ce milieu dans le traitement de maladies
US10874699B2 (en) 2013-07-05 2020-12-29 Université Catholique de Louvain Conditioned medium from human adult liver stem cells and its use in the treatment of liver disorders
US11793837B2 (en) 2013-07-05 2023-10-24 Université Catholique de Louvain Conditioned medium from human adult liver stem cells and its use in the treatment of liver disorders
US10744160B2 (en) 2014-12-01 2020-08-18 T-Helper Cell Technologies, Llc Stem cell material and method of manufacturing
US11376283B2 (en) 2016-05-31 2022-07-05 T-Helper Cell Technologies, Llc Stem cell material, compositions, and methods of use
US20200157500A1 (en) * 2017-06-30 2020-05-21 Cha Biotech Co., Ltd. Stem cell-derived skin precursor cell culture medium and preparation method therefor
US11773370B2 (en) * 2017-06-30 2023-10-03 Cha Biotech Co., Ltd. Stem cell-derived skin precursor cell culture medium and preparation method therefor

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