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WO2010059862A2 - Compositions et procédés de génération d’unités pilo-sébacées - Google Patents

Compositions et procédés de génération d’unités pilo-sébacées Download PDF

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Publication number
WO2010059862A2
WO2010059862A2 PCT/US2009/065202 US2009065202W WO2010059862A2 WO 2010059862 A2 WO2010059862 A2 WO 2010059862A2 US 2009065202 W US2009065202 W US 2009065202W WO 2010059862 A2 WO2010059862 A2 WO 2010059862A2
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WO
WIPO (PCT)
Prior art keywords
cells
composition
hair
dermal
skin
Prior art date
Application number
PCT/US2009/065202
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English (en)
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WO2010059862A3 (fr
Inventor
Lily Lee
Cheng Ming Chuong
Warren Garner
Ting Xin Jiang
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University Of Southern California
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Priority to US13/130,296 priority Critical patent/US20110321180A1/en
Publication of WO2010059862A2 publication Critical patent/WO2010059862A2/fr
Publication of WO2010059862A3 publication Critical patent/WO2010059862A3/fr

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    • 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
    • C12N5/0697—Artificial constructs associating cells of different lineages, e.g. tissue equivalents
    • C12N5/0698—Skin equivalents
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    • 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
    • C12N5/0625—Epidermal cells, skin cells; Cells of the oral mucosa
    • C12N5/0627—Hair cells
    • C12N5/0628—Hair stem cells; Hair progenitors
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    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/05—Inorganic components
    • C12N2500/10—Metals; Metal chelators
    • C12N2500/20—Transition metals
    • C12N2500/24—Iron; Fe chelators; Transferrin
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    • C12N2500/90—Serum-free medium, which may still contain naturally-sourced components
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/11—Epidermal growth factor [EGF]
    • C—CHEMISTRY; METALLURGY
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/135—Platelet-derived growth factor [PDGF]
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/155—Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/165—Vascular endothelial growth factor [VEGF]
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/30—Hormones
    • C12N2501/38—Hormones with nuclear receptors
    • C12N2501/385—Hormones with nuclear receptors of the family of the retinoic acid recptor, e.g. RAR, RXR; Peroxisome proliferator-activated receptor [PPAR]
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/999—Small molecules not provided for elsewhere
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    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/09—Coculture with; Conditioned medium produced by epidermal cells, skin cells, oral mucosa cells
    • C12N2502/092—Coculture with; Conditioned medium produced by epidermal cells, skin cells, oral mucosa cells hair cells
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    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/09—Coculture with; Conditioned medium produced by epidermal cells, skin cells, oral mucosa cells
    • C12N2502/094—Coculture with; Conditioned medium produced by epidermal cells, skin cells, oral mucosa cells keratinocytes
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    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/13—Coculture with; Conditioned medium produced by connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
    • C12N2502/1323—Adult fibroblasts
    • C—CHEMISTRY; METALLURGY
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    • C12N2503/00—Use of cells in diagnostics
    • C12N2503/04—Screening or testing on artificial tissues
    • C12N2503/06—Screening or testing on artificial skin
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    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/50—Proteins
    • C12N2533/54—Collagen; Gelatin

Definitions

  • Figure 10 illustrates the composition of a viral vector which can be used in combination with packaging and pseudotyping construct to generate lentivirus useful for molecular reprogramming.
  • stem cell defines a cell with the ability to divide for indefinite periods in culture and give rise to specialized cells.
  • stem cells are categorized as somatic (adult) or embryonic.
  • a somatic stem cell is an undifferentiated cell found in a differentiated tissue that can renew itself (clonal) and (with certain limitations) differentiate to yield all the specialized cell types of the tissue from which it originated.
  • An embryonic stem cell is a primitive (undifferentiated) cell from the embryo that has the potential to become a wide variety of specialized cell types.
  • An embryonic stem cell is one that has been cultured under in vitro conditions that allow proliferation without differentiation for months to years.
  • progenitor cells include, but are not limited to, satellite cells found in muscles, intermediate progenitor cells formed in the subventhcular zone, bone marrow stromal cells, periosteum progenitor cells, pancreatic progenitor cells and angioblasts or endothelial progenitor cells.
  • progenitor cells may also include, but are not limited to, epidermal and dermal cells from neonatal organisms.
  • Proteins that positively or negatively regulate the BMP signaling are also considered within the meaning of the BMP signaling. Proteins that positively regulate BMP signaling include, but are not limited to, Serine/threonine-protein kinase receptor R3 (ACVRL1 , UniProt: P37023) and Endoglin (ENG, UniProt: P17813).
  • ACVRL1 Serine/threonine-protein kinase receptor R3
  • Endoglin ENG, UniProt: P17813
  • An "agonist”, as used herein, refers to a drug or other chemical that can bind a receptor on a cell to produce a physiologic reaction typical of a naturally occurring substance. The efficacy of an agonist may be positive, causing an increase in the receptor's activity or negative causing a decrease in the receptor's activity.
  • An "antagonist” refers to a type of receptor ligand or drug that does not provoke a biological response itself upon binding to the receptor, but blocks or dampens agonist-mediated responses. In pharmacology, antagonists have affinity but no efficacy for their cognate receptors and binding will disrupt the interaction and inhibit the function of an agonist or inverse agonist at receptors.
  • Antagonists mediate their effects by binding to the active site or to allosteric sites on receptors or they may interact at unique binding sites not normally involved in the biological regulation of the receptor's activity. Antagonist activity may be reversible or irreversible depending on the longevity of the antagonist-receptor complex which in turn depends on the nature of antagonist receptor binding. The majority of drug antagonists achieve their potency by competing with endogenous ligands or substrates at structurally defined binding sites on receptors.
  • hair growth intends to include, but not limited to, the formation of new hair or growth of existing hair.
  • Spironolactone (IUPAC name: 7 ⁇ -Acetylthio-3-oxo-17 ⁇ -pregn-4-ene- 21 ,17-carbolactone is marketed under the trade names Aldactone, Novo-Spiroton, Aldactazide, Spiractin, Spirotone, Verospiron or Berlactone) is a diuretic and is used as an antiandrogen. It is also used for treating hair loss in women, and can be used as a topical medication for treatment of male baldness.
  • Minoxidil (trade names Rogaine and Regaine; IUPAC name: 6-piperidin- 1 -ylpyhmidine-2,4-diamine 3-oxide) is a commercially available topical formulation that inhibits hair loss, is a vasodilator medication that is available over the counter for treatment of androgenic alopecia, among other baldness treatments.
  • Finasteride (IUPAC name ⁇ /-(1 ,1-dimethylethyl)-3-oxo-(5 ⁇ ,17 ⁇ )-4- azaandrost-1 -ene-17-carboxamide) is a synthetic antiandrogen that acts by inhibiting type Il 5-alpha reductase, the enzyme that converts testosterone to dihydrotestosterone (DHT). It is used to treat prostate cancer and is registered in many countries to treat adrogenetic alopecia or male pattern baldness.
  • DHT dihydrotestosterone
  • Propecia is a medicament containing finasteride as an active ingredient is commercially available from Merck & Co., Inc.
  • administering refers to the delivery of a medication or matrix composition to a mammal or subject to be treated and/or in need of such treatment.
  • Non-limiting examples include oral dosing, intracutaneous injection, direct application to target area proximal areas on the skin, or applied on a patch.
  • Various physical and/or mechanical technologies are available to permit the sustained or immediate topical or transdermal administration of macromolecules (such as, peptides). Such technologies include iontophoresis (see for example KaNa et al. (2004) Adv. Drug Del. Rev.
  • microstructured arrays sometimes called microneedles; one particular example is the Microstructured Transdermal System (MTS) commercially available from 3M) (see, e.g., Alain et al. (2002) J. Control. Release 81 :113-119; Santi et al. (1997) Pharm. Res. 14(1 ):63-66; Sebastien et al. (1998) J. Pharm. Sci. 87(8):922-925).
  • Methods of making and using arrays of solid microneedles that can be inserted into the skin for transdermal delivery of peptides are provided in Martanto et al.
  • the delivery system includes a combination of systems, such as microneedles made of biocompatible and biodegradable polymers (Park et al. (2005) J. Control. Release 104:51 -66).
  • administration is topical administration as defined herein.
  • Topical administration refers to delivery of a composition or medication by application to the skin.
  • Non-limiting examples of topical administration include any methods described under the definition of "administration” pertaining to delivery of a medication to the skin.
  • the pharmaceutically acceptable carrier facilitate immediate or controlled release of the active ingredient.
  • this invention provides a composition useful in one aspect to generate pilosebaceous units in a physiological plane comprising, or alternatively consisting essentially of, or yet further consisting of, a biocompatible scaffold and an effective amount of skin precursor cells contained within or on the matrix.
  • the skin precursor cells comprise, or alternatively consist essentially of, or yet further consist of, stem cells, epidermal precursor cells or dermal precursor cells.
  • Stem cells, epidermal and/or dermal precursor cells can be of any appropriate type, e.g., an animal such as a mammal, including a human.
  • Non-human animals include, for example, murine (such as rats or mice), canine, such as dogs, leporids, such as rabbits, equine, bovine, simian, livestock, sport animals, and pets.
  • the cell species type is selected for compatibility with the host into which the composition is implanted, e.g., murine for a murine host and human for a human host.
  • adult or somatic stem cells can be utilized in the compositions of this invention.
  • the cells are identified by the stem cells markers and can be isolated using the methods as described by, e.g., Reiisi (2009) In Vitro Cell Dev. Biol. Anim. 2009 Nov 14 [Epub ahead of print].
  • compositions comprises, or alternatively consists essentially of or yet further consists of a combination of dermal precursor cells, epidermal precursor cells and stem cells, e.g. one or more of adult or somatic stem cells, embryonic stem cells and iPS cells.
  • the cells are combined with a biocompatible matrix.
  • a biocompatible matrix As used herein, the term intends a compositions that has the ability to support cell growth either in vitro or in vivo, the ability to support the growth of pilosebaceous units, the ability to be endowed with varying degrees of flexibility or rigidity required, the ability to have varying degrees of biodegradability, the ability to be introduced into the intended site in vivo without provoking secondary damage, and the ability to serve as a vehicle or reservoir for delivery of drugs or bioactive substances to the desired site of action.
  • Prior art matrices are known, and include for example gels, foams, sheets, and numerous porous particulate structures of different forms and shapes.
  • the matrix can be composed of biopolymers, including polypeptides or proteins, as well as various polysaccharides, including proteoglycans and the like.
  • these biopolymers may be either selected or manipulated in ways that affect their physico-chemical properties.
  • biopolymers may be cross- linked either enzymatically, chemically or by other means, thereby providing greater or lesser degrees of rigidity or susceptibility to degradation.
  • Natural polymers which have been disclosed to be useful for tissue engineering or culture, one can enumerate various constituents of the extracellular matrix including hydrogels, fibronectin, various types of collagen, and laminin, as well as keratin, fibrin and fibrinogen, hyaluronic acid, heparin sulfate, chondroitin sulfate and others. These are described for example in U.S. Patent Publ. No. 2005/0260753, U.S. Patent Nos.
  • a biocompatible matrix includes acellular matrices that have hair enhancing activity which can be prepared according to methods described in the literature, e.g., Schedin et al. (2004) Oncogene. 23(9):1766-79 and Potapova et al. (2008) Am. J. Physiol. Heart Circ. Physiol. 295(6):H2257-63, from E13 mouse dermis which has hair enhancing ability.
  • the composition further comprises a detectable marker or label to monitor growth and differentiation of the cells.
  • a detectable marker or label to monitor growth and differentiation of the cells.
  • examples of such include for example, luciferase under the control of a ubiquitin promoter, GFP, herpes simplex virus type 1 thymidine kinase (HSV-1 TK) under the control of a ubiquitin promoter and super-paramegnetic iron oxide (SPIO) nanoparticles.
  • luciferase under the control of a ubiquitin promoter
  • GFP herpes simplex virus type 1 thymidine kinase
  • SPIO super-paramegnetic iron oxide
  • compositions can alternatively contain an effective amount of differentiation or growth factor that promotes cell differentiation or growth.
  • growth factors include agents that inhibit Born Morphogenic Protein (BMP) signaling, such as noggin (UniProt: Q13253) which can be produced using methods described in, e.g. McMahon et al. (1998) Genes & Development, 12:1438-52, chordin, gremlin, sclerostin and follistatin and any combination thereof.
  • BMP Born Morphogenic Protein
  • noggin UniProt: Q13253
  • Use of the terms such as "growth factors, cytokines, hormones” is to be exemplary.
  • the factor comprises Platelet Derived Growth Factor (PDGF) available from R&D Ssytems, Minneapolis, MN, Vascular Endothelial Growth Factor (VEGF) available from Abeam, Cambridge, MA, Epithelial Growth Factor (EGF) available from Abeam, Cambridge, MA, TGF- ⁇ available from Abeam, Cambridge, MA, Fibroblast Growth Factor (FGF), insulin available from Abeam, Cambridge, MA, transferrin, retinoid, or any combination thereof.
  • PDGF Platelet Derived Growth Factor
  • VEGF Vascular Endothelial Growth Factor
  • EGF Epithelial Growth Factor
  • TGF- ⁇ available from Abeam, Cambridge, MA
  • FGF Fibroblast Growth Factor
  • insulin available from Abeam, Cambridge, MA, transferrin, retinoid, or any combination thereof.
  • the composition is suitable for culturing mammalian epidermal cells and therefore can comprise cell culture medium as known to those of skill in the art, e.g., without limitation serum-free medium commercially available from Invitrogen (Carlsbad, California). Additional components are optionally added to the composition, that include, but are not limited to antibiotics, albumin, amino acids, and other components known to the art for the culture of cells. Additionally, components optionally are added to enhance the differentiation process. Effective amounts of the differentiation and/or growth factors can be empirically determined by those of skill in the art.
  • compositions can alternatively contain an effective amount of minoxidil (commercially available under the trademark "Rogaine” (Pharmacia & Upjohn Company)), finasteride or other agent that enhances hair growth.
  • minoxidil commercially available under the trademark "Rogaine” (Pharmacia & Upjohn Company)
  • finasteride or other agent that enhances hair growth.
  • the invention also provides compositions comprising the serum-free medium described above, wherein the medium comprises reduced concentrations of one or more factors that modulate cell growth.
  • the factor comprises PDGF, VEGF, EGF, TGF- ⁇ , FGF, insulin, transferrin, retinoid,, or any combination thereof.
  • the medium is suitable for cultuhng mammalian (e.g., murine, rat or human) epidermal cells.
  • the culturing comprises cell differentiation.
  • the epidermal cells comprise keratinocyte stem cells, follicular papillae, sheath cells, non-stem cell keratinocytes, or any combination thereof.
  • the concentration of cells in the scaffold is from about 800,000 cells/mm 3 to about 1 ,500,000 cells/mm 3 .
  • the concentration of cells in the scaffold is equal to or greater than about 10,000 cells/mm 3 , or alternatively is equal to or greater than about 50,000 cells/mm 3 , or alternatively is equal to or greater than about 100,000 cells/mm 3 , or alternatively is equal to or greater than about 200,000 cells/mm 3 , or alternatively is equal to or greater than about 300,000 cells/mm 3 , or alternatively is equal to or greater than about 400,000 cells/mm 3 , or alternatively is equal to or greater than about 500,000 cells/mm 3 , or alternatively is equal to or greater than about 600,000 cells/mm 3 , or alternatively is equal to or greater than about 700,000 cells/mm 3 , or alternatively is equal to or greater than about 800,000 cells/mm 3 , or alternatively is equal to or greater than about 900,000 cells/mm 3 , or or alternatively is equal to or greater than about 10,000 cells/mm 3
  • the concentration of cells in the scaffold is equal to or less than about 800,000 cells/mm 3 , or alternatively is equal to or less than about 900,000 cells/mm 3 , or alternatively is equal to or less than about 1 ,000,000 cells/mm 3 , or alternatively is equal to or less than about 1 ,100,000 cells/mm 3 , or alternatively is equal to or less than about 1 ,200,000 cells/mm 3 , or alternatively is equal to or less than about 1 ,300,000 cells/mm 3 , or alternatively is equal to or less than about 1 ,400,000 cells/mm 3 , or alternatively is equal to or less than about 1 ,500,000 cells/mm 3 , or alternatively is equal to or less than about 1 ,600,000 cells/mm 3 , or alternatively is equal to or less than about 1 ,700,000 cells/mm 3 , or alternatively is equal to or less than about 1 ,800,000 cells/mm 3 , or alternatively is equal to or less than about 1 ,900,000 cells/mm
  • the skin precursor cells comprise epidermal and dermal precursor cells.
  • the ratio of epidermal to dermal precursor cells is about 2 : 1 , or alternatively about 1 : 1 , or alternatively about 1 : 2, or alternatively about 1 : 3, or alternatively about 1 : 4, or alternatively about 1 : 5, or alternatively about 1 : 6, or alternatively about 1 : 7, or alternatively about 1 : 8, or alternatively about 1 : 9, or alternatively about 1 : 10, or alternatively about 1 : 12, or alternatively about 1 : 15, or alternatively about 1 : 20 or alternatively about 1 : 50.
  • the composition can further comprise, or alternatively consist essentially of, or yet further consist of, an effective amount of a suitable carrier and/or a growth or differentiation factor.
  • the factor is selected from the group consisting of noggin, chordin, gremlin, sclerostin and follistatin and combinations thereof.
  • the factor is selected from the group consisting of Platelet Derived Growth Factor (PDGF), Vascular Endothelial Growth Factor (VEGF), Epithelial Growth Factor (EGF), TGF- ⁇ , Fibroblast Growth Factor (FGF), insulin, transferrin, retinoid and combinations thereof.
  • the composition further comprises a detectable marker or label to monitor growth and differentiation of the cells.
  • a detectable marker or label to monitor growth and differentiation of the cells.
  • examples of such include for example, luciferase under the control of a ubiquitin promoter, GFP, herpes simplex virus type 1 thymidine kinase (HSV-1 TK) under the control of a ubiquitin promoter and super-paramegnetic iron oxide (SPIO) nanoparticles.
  • luciferase under the control of a ubiquitin promoter
  • GFP herpes simplex virus type 1 thymidine kinase
  • SPIO super-paramegnetic iron oxide
  • the composition may be prepared by admixing an effective amount of isolated skin precursor cells in serum-free medium and a biocompatible scaffold, under conditions that favor the incorporation of the cells into the biocompatible scaffold.
  • serum-free media can support the maintenance and expansion of stem cells or precursor cells and various types of serum-free medic are commercially available from vendors.
  • StemSpan® SFEM and StemSpan® H3000 are available from STEMCELL Technologies, Vancouver, BC, Canada.
  • the concentration of the cells to be admixed with the scaffold is an amount that will produce a concentration in the medium from about 800,000 cells/mm 3 to about 1 ,500,000 cells/mm 3 .
  • the scaffold and cells are admixed by passively contacting the cells with the scaffold at a temperature range from about 25 to about 37 0 C for about 30 minutes to about 2 hours.
  • the media containing the cells is merely placed on a surface of the scaffold.
  • different ratios between the epidermal and dermal populations can be used to make the composition.
  • Table 1 a ratio of epidermal and dermal precursor cells between about 1 : 5 and about 1 : 10 can be used to generate good pilosebaceous units.
  • a combination of aged epidermal cells and newborn dermal cells, or a combination of newborn epidermal cells and aged dermal cells may not give rise to good hair growth.
  • Precursor cells can also be used to generate good hair growth.
  • a combination of positive precursor cells and whole skin (WT) cells can lead to fair hair growth as well. While the use of Integra Matrix produced good hair growth, use of other scaffolds can result in good hair growth too.
  • This invention further provides a dermal patch comprising the compositions as noted above in combination with a dressing.
  • a "dressing" refers to an overlay adjunct used by a mammal for application to a wound to promote healing and/or prevent further harm.
  • a dressing may further comprise a bandage, which is primarily used to hold a dressing in place.
  • a dressing can control the moisture content, protect the wound from infection, remove slough, or maintain the optimum pH or temperature to encourage healing.
  • Non-limiting examples of dressings include a silicone protective layer or sheet, a collagen sheet, a plastic sheet or a latex sheet.
  • the dressing is sterile.
  • the surface area of the dressing includes the entire area of the patch and extends beyond the periphery of the dermal patch and may optionally include an adhesive layer or coating around the periphery of the dressing but excluding the area of the patch.
  • the adhesive coating or layer serves to secure the dermal patch to the situs of application.
  • the adhesive coating may exclude or include the area of the patch and if the adhesive coating includes the area of patch then the adhesive coating is irreversibly attached to the patch, or the adhesive coating can be reversible.
  • the dermal patch is stably attached to the dressing.
  • the dermal patch is removably attached to the dressing to allow for removing the patch overlay without removing the underlying patch.
  • Also provided by this invention is a method for generating pilosebaceous units in a physiological plane in a mammal in need thereof, comprising implanting the composition of the invention into the dermal layer of the subject such as a mammal under conditions that favor implantation of the composition into the dermis of the mammal.
  • mammals include, but are not limited to, murines, rats, simians, bovines, canines, humans, farm animals, sport animals and pets.
  • biocompatible scaffolds for use in this invention include, but are not limited to the porous and/or biodegradable and/or biocompatible scaffold as described in U.S. Patent No. 4,947,840, col. 2, line 27 to col. 5, line 10, incorporated herein by reference in its entirety.
  • a biocompatible scaffold is a dermal substitute consisting of amnion and biodegradable polymer as described in U.S. Patent Application Publication No. US 2005/0107876, paragraphs 28 to 64.
  • a biocompatible scaffold is a single or double density biopolymer foam as described in International Patent Application Publication No. WO 98/22154, page 5, line 32 to page 23, line 33.
  • a biocompatible scaffold is a gel-matrix-cells integrated system as described in International Patent Application Publication No. WO 2007/141028, page 13, line 1 to page 21 , line 2.
  • a biocompatible scaffold is a biomechanical implant as described in International Patent Application Publication No. WO 98/40111 , page 7, line 13 to page 19, line 9.
  • biocompatible scaffolds include, but are not limited to, Alloderm dermal collagen matrix (LifeCell Corporation, Branchburg, NJ), Dermagraft-TC woven bioabsorbable polymer (polyglycolic and polylactic acids) membrane (Advanced Tissue Sciences, La JoIIa, Calif),
  • the biocompatible scaffold can be type I collagen or silicon cell culture insert which are commercially available (eg. FalconTM Cell Culture Insert from BD Biosciences, San Jose, CA).
  • the resulted concentration of cells in the scaffold is from about 800,000 cells/mm 3 to about 1 ,500,000 cells/mm 3 .
  • the concentration of cells in the scaffold is equal to or greater than about 10,000 cells/mm 3 , or alternatively is equal to or greater than about 50,000 cells/mm 3 , or alternatively is equal to or greater than about 100,000 cells/mm 3 , or alternatively is equal to or greater than about 200,000 cells/mm 3 , or alternatively is equal to or greater than about 300,000 cells/mm 3 , or alternatively is equal to or greater than about 400,000 cells/mm 3 , or alternatively is equal to or greater than about 500,000 cells/mm 3 , or alternatively is equal to or greater than about 600,000 cells/mm 3 , or alternatively is equal to or greater than about 700,000 cells/mm 3 , or alternatively is equal to or greater than about 800,000 cells/mm 3 , or alternatively is equal to or greater than about 900,000 cells/mm 3 , or alternatively is equal to or greater than about 1 ,000,000 cells/mm 3 .
  • the skin precursor cells comprise epidermal and dermal precursor cells.
  • the ratio of epidermal to dermal precursor cells is about 2 : 1 , or alternatively about 1 : 1 , or alternatively about 1 : 2, or alternatively about 1 : 3, or alternatively about 1 : 4, or alternatively about 1 : 5, or alternatively about 1 : 6, or alternatively about 1 : 7, or alternatively about 1 : 8, or alternatively about 1 : 9, or alternatively about 1 : 10, or alternatively about 1 : 12, or alternatively about 1 : 15, or alternatively about 1 : 20 or alternatively about 1 : 50.
  • the admixing is performed by passively contacting the cells with the scaffold, such as by soaking the scaffold with the cell composition in a pharmaceutically acceptable carrier at a temperature of about 25 to about 37°C.
  • the biocompatible scaffold is dried or lyophilized prior to admixing with the cells in serum-free medium.
  • the method comprises, or alternatively consists essentially of, or alternatively, consists of admixing an effective amount of a growth factor selected from the group consisting of Platelet Derived Growth Factor (PDGF), Vascular Endothelial Growth Factor (VEGF), Epithelial Growth Factor (EGF), TGF- ⁇ , Fibroblast Growth Factor (FGF), insulin, transferrin, retinoid and combinations thereof.
  • PDGF Platelet Derived Growth Factor
  • VEGF Vascular Endothelial Growth Factor
  • EGF Epithelial Growth Factor
  • TGF- ⁇ TGF- ⁇
  • Fibroblast Growth Factor FGF
  • insulin transferrin, retinoid and combinations thereof.
  • the skin precursor cells comprise dermal and epidermal precursor cells.
  • the precursor cells comprise progenitor cells from adult skin or other tissues containing stem cells.
  • the precursor cells can be adult or embryonic stem cells having the ability to differentiate into hair follicles under appropriate culturing or growth conditions that are present in the micro- or macro-environment (see e.g. Yu et al. (2006) Am. J. Pathol. 168(6):1979-88).
  • the skin precursor cells are embryonic stem (ES) cells.
  • ES cells have the potential to develop into different cell types. Attempts have been made to guide them toward a particular lineage with selected medium conditions, activating endogenous transcriptional factors (Pera & Trounson (2004) Development 131 (22):5515-25), transfecting cells with specific transcriptional factors (Muller et al. (2000) FASEB J. 14(15):2540-8), or co-culturing them with cells capable of lineage induction (Kawasaki et al. (2000) Neuron 28(1 ):31 -40).
  • Several successful methods can guide mouse ES cells toward a keratinocyte lineage (Aberdam (2004) Int. J. Dev. Biol.
  • the skin precursor cells are cells isolated from human tissues.
  • the skin precursor cells are Foreskin cells isolated from young children.
  • the precursor cells are from adult human tissues.
  • the skin precursor cells are cells isolated from the patient in need of the treatment.
  • One of the risks in using stem cells is immunologic rejection, which can be alleviated by using a patient's own cells. It is valuable to isolate or convert adult cells to multi-potential skin stem cells.
  • the skin precursor cells can be isolated from adult mammalian skin, such as Skin-derived Precursors (SKP) cells (Toma et al.
  • SKP Skin-derived Precursors
  • These adult cells can be converted or differentiated into hair forming cells with procedures described in e.g. Hunt et al. (2008) Stem Cells 26(1 ):163-72.
  • small molecules such as those targeting genes in the BMP pathway and Wnt pathway may be used to convert adult skin cells into progenitor cells (see e.g. Plikus et al. (2008) Nature 451 (17):340-345).
  • acellular matrix is used.
  • Acellular matrix is prepared from mammalian tissues (Schedin et al. (2004) Oncogene. 23(9):1766-79; Potapova et al. (2008) Am. J. Physiol. Heart Circ. Physiol. 295(6):H2257-63).
  • Candidate cells are seeded in an acellular matrix derived from E13 mouse skin which has strong hair inducing ability. Hair follicles can induced from proper candidate cells.
  • small molecules and growth factors are used to induce the hair forming capability in cells. These cells are pretreated with growth factors or small molecules. Selection of candidate growth factors or small molecules is based on literature or microarray gene profiling analysis. They can be tested with methods described herein.
  • the skin precursor cells are Induced Pluhpotent Stem (iPS) cells generated from cells isolated from adult tissues such as the skin by altering the transcription profile in the adult cells (see Takahashi et al. (2007) Cell 131 (5):861 -872 and Yu et al. (2007) Science 318(5858):1917-1920).
  • iPS cells can be converted to hair forming dermal papilla when they are incubated with stem cells with hair forming epidermis.
  • the iPS cells can be converted to hair forming epidermis when they are incubated with stem cells with hair forming dermis or cell free matrix.
  • the skin precursor cells comprise human adult keratinocytes and fibroblast cells.
  • Sources of skin precursor cells can be tested experimentally.
  • multi-potential epidermal or dermal stem cells can be tested experimentally.
  • newborn mouse skin cells can serve as the positive control.
  • Human or mouse or other types of mammalian epidermal stem cell candidates are tested in combination with newborn mouse dermal cells.
  • Human or mouse or other types of mammalian dermal stem cell candidates are tested in combination with newborn mouse epidermal cells.
  • Candidate cells are evaluated with a three-tier assay system with a higher throughput type screening first, and then with two of the more rigorous tests for hair forming ability.
  • Tier (i) Mixed aggregate assay. In this assay, tested cells are mixed and cultured in shaking gassed flasks. Cells interact and differentiation genes are induced when right interactions occur.
  • Tier (ii) Patch assay (as described in Zheng et al. (2005) J. Invest. Dermatol. 124(5):867-876). In this assay, dermal and epidermal cells are mixed in a high density suspension and injected subcutaneously. This assay is used to test the ability of cells to form a hair follicle structure.
  • Tier (iii) Planar hair forming assay evaluates the topology of the whole hair follicle population to see if they are properly or physiologically arranged.
  • the composition may be prepared by admixing an effective amount of isolated skin precursor cells in serum-free medium or other pharmaceutically acceptable carrier and a biocompatible scaffold, under conditions that favor the incorporation of the cells into the biocompatible scaffold.
  • the scaffold and cells are admixed by passively contacting the cells with the scaffold at a temperature range from about 25 to about 37 0 C for about 30 minutes to about 2 hours. Passive is just applying to the surface of the scaffold. Additional agents, as describe above, can be further combined with the cells and scaffold.
  • Another aspect of this invention provides a method for generating pilosebaceous units in a physiological plane in a mammal in need thereof, comprising implanting the composition of this invention into the dermal layer of the mammal under conditions that favor implantation of the composition into the dermis of the mammal.
  • the resulted concentration of cells in the scaffold is from about 800,000 cells/mm 3 to about 1 ,500,000 cells/mm 3 .
  • the concentration of cells in the scaffold is equal to or greater than about 10,000 cells/mm 3 , or alternatively is equal to or greater than about 50,000 cells/mm 3 , alternatively is equal to or greater than about 100,000 cells/mm 3 , alternatively is equal to or greater than about 200,000 cells/mm 3 , alternatively is equal to or greater than about 300,000 cells/mm 3 , alternatively is equal to or greater than about 400,000 cells/mm 3 , alternatively is equal to or greater than about 500,000 cells/mm 3 , alternatively is equal to or greater than about 600,000 cells/mm 3 , alternatively is equal to or greater than about 700,000 cells/mm 3 , alternatively is equal to or greater than about 800,000 cells/mm 3 , alternatively is equal to or greater than about 900,000 cells/mm 3 , or alternatively is equal to or greater than about 1 ,000,000 cells/mm 3 .
  • the concentration of cells in the scaffold is equal to or less than about 800,000 cells/mm 3 , or alternatively is equal to or less than about 900,000 cells/mm 3 , or alternatively is equal to or less than about 1 ,000,000 cells/mm 3 , or alternatively is equal to or less than about 1 ,100,000 cells/mm 3 , or alternatively is equal to or less than about 1 ,200,000 cells/mm 3 , or alternatively is equal to or less than about 1 ,300,000 cells/mm 3 , or alternatively is equal to or less than about 1 ,400,000 cells/mm 3 , or alternatively is equal to or less than about 1 ,500,000 cells/mm 3 , or alternatively is equal to or less than about 1 ,600,000 cells/mm 3 , or alternatively is equal to or less than about 1 ,700,000 cells/mm 3 , or alternatively is equal to or less than about 1 ,800,000 cells/mm 3 , or alternatively is equal to or less than about 1 ,900,000 cells/mm
  • the skin precursor cells comprise epidermal and dermal precursor cells.
  • the ratio of epidermal to dermal precursor cells is about 2 : 1 , or alternatively is about 1 : 1 , or alternatively is about 1 : 2, or alternatively is about 1 : 3, or alternatively is about 1 : 4, or alternatively is about 1 : 5, or alternatively is about 1 : 6, or alternatively is about 1 : 7, or alternatively is about 1 : 8, or alternatively is about 1 : 9, or alternatively is about 1 : 10, or alternatively is about 1 : 12, or alternatively is about 1 : 15, or alternatively is about 1 : 20 or alternatively is about 1 : 50.
  • Animals can also be engineered to express GFP constitutively. Transplantation of these cells onto a GFP negative host will allow one to visualize the organization of GFP positive cells using fluorescent microscopy (e.g. Leica Z16 APO fluorescent microscope). While the resolution of fluorescent imaging is much better, the light penetration is not good.
  • fluorescent microscopy e.g. Leica Z16 APO fluorescent microscope
  • ultrasound can be used for tracking the cells.
  • the skin is first shaved (not plucked) to avoid any damage to the hair follicles.
  • the skin is then covered with aquasonic gel to facilitate contact of the ultrasound probe. Images are videotaped to produce real time movies of the skin. The overall architecture can be visualized.
  • the invention provides a method for generating pilosebaceous units in a physiological plane in a mammal in need thereof, comprising, or alternatively consisting essentially of, or yet further consisting of implanting the composition of the invention into the dermal layer of the mammal under conditions that favor implantation of the composition into the dermis of the mammal.
  • the composition replaces the entire skin within the area.
  • the epidermis and all of the dermis are replaced by the composition.
  • the epidermis and part of the dermis are replaced by the composition.
  • the invention provides a method for preparing pilosebaceous units in a physiological plane, comprising admixing skin precursor cells and a medium, wherein the concentration of skin precursor cells present in the medium is greater than about 1 x 10 7 cells per milliliter of medium. In some embodiments, the concentration of skin precursor cells present in the medium is less than about 1 x 10 8 cells per milliliter of medium.
  • the method further comprises, or alternatively consists essentially of, or yet further consists of, the step of overlaying an epithelial sheet on the admixed dermal precursor cells and the medium.
  • the pilosebaceous units prepared by the method of the invention can be used to treat a condition in a mammalian subject in need of, which condition comprises hair loss or insufficient hair growth.
  • the condition is alopecia.
  • the condition is wound healing.
  • human cell sources may be implanted into mice (nude mice as shown below) and then agents are contacted with the implant either by incorporation into the matrix or alternatively by subsequent administration to the implanted cells and growth is monitored.
  • the test agents can be compared to known agents that modulate hair growth, for example noggin or MinoxidolTM to determine if they are candidate leads for further development. This is a fast and simple clinically relevant animal model for high-throughput screening of various test agents.
  • agents, compositions and methods of the present invention in any of the above embodiments can be used in the manufacture of medicaments and for the treatment of humans and other animals by administration in accordance with conventional procedures, such as an active ingredient in pharmaceutical compositions.
  • Multipotential skin precursor cells are currently obtained from neonatal mice using techniques from previously published work (see e.g. Lichit et al. (1993) J. Invest. Dermatol. 101 (1 Suppl):124S-129S). Briefly, neonatal mice are harvested shortly after birth (within the first 24 hours) and euthanized.
  • the in vivo graft bed is prepared using sterile technique. A full thickness piece of skin, approximately the size of the collagen matrix, is excised. Once bleeding has been controlled, the collagen matrix or silicone cell insert, with the cells seeded inside or on top, is grafted by suture ligation such that the cells are against the wound bed with a silicone protective layer level with the epidermis. Sterile dressing are applied in order to provide constant pressure against the graft to the wound bed so that the graft has the best chance of capillary formation and being incorporated as part of the host's skin. Dressings are then taken down for inspection of the wound on days 6-8 post graft.
  • This method is highly reproducibly that cells can be easily grafted with the help of a scaffold-like matrix to allow cells to reorganize and grow new hair in a cosmetically acceptable fashion. Hairs have been allowed to grow up to 3 months, giving evidence that the hair is permanent and able to cycle normally.
  • GFP positive precursor cells also showed good hair growth.
  • a combination GFP positive precursor cells and whole skin (WT) cells led to fair hair growth as well. While the use of Integra Matrix produced good hair growth, use of other scaffold such as type I collagen resulted in mostly good hair growth too.
  • GFP WT 2 0 2 0
  • This invention provides a procedure that allows precursor cells to self-organize in order to generate a large number of new hair follicles which are arranged in a plane with a cosmetically acceptable appearance.
  • the formed hairs are able to cycle through the normal hair cycles of regeneration.
  • This procedure can be performed efficiently, reproducibly and on a large scale so that clinical applications can be envisioned.
  • the hair grows in a topologically proper environment such that there is room for cycling and hairs to fall out and regrow. This technology is useful to form skin with hairs on patients suffering from severe injuries such as burn or alopecia patients.
  • dorsomorphin is a potent small molecule BMP antagonist (Hao et al. (2008) PLoS ONE 3(8):e2904, Yu et al. (2008) Nat. Chem. Biol. 4(1 ):33-41 ).
  • Dorsomorphin was reported to selectively inhibit the BMP receptors, type I: ALK2, ALK3 and ALK6 and thus "blocks BMP-mediated SMAD1/5/8 phosphorylation".
  • Dorsomorphin has preferential specificity toward inhibiting BMP vs.
  • Dorsomorphin is characterized by low toxicity.
  • Dorsomorphin is currently commercially available from several vendors.
  • Dorsomorphin can be delivered into skin to lower macro-environmental BMP signaling and create favorable conditions for hair growth to occur.
  • Multipotential skin precursor cells are currently obtained from neonatal mice using techniques from previously published work. Briefly, neonatal mice are harvested shortly after birth (within the first 24 hours) and euthanized. The truncal skin is then dissected off with sharp forceps. Epidermis and dermis are separated by floating in cold 0.25% trypsin overnight. Epidermal cells are then dissociated into a cell suspension by cutting into fine pieces and manual tituration with a serological pipet. Single epithelial cells are separated through a 70 ⁇ m cell strainer to exclude cells of the stratum corneum. The dermal cells are individually dissociated using warm 0.35% collagenase for 40-50 minutes at 37°C.
  • Skin precursor cells can also be seeded into a specially prepared matrix which may enhance their hair-forming ability or provide better maneuverability.
  • An acellular matrix can be prepared from E13 mouse dermis which has a high hair- inducing ability (Schedin et al. (2004) Oncogene. 23(9):1766-79; Potapova et al. (2008) Am. J. Physiol. Heart Circ. Physiol. 295(6):H2257-63).
  • the procedure is flexible and allows one to shape reconstituted hair regions as may be needed for adding an eye brow or scalp hairs.
  • the region can also be very small ( ⁇ 5 X 5mm), making it feasible for alopecia patients.

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Abstract

L'invention porte sur des compositions et des procédés de génération d’unités pilo-sébacées. Sous un aspect, l'invention comporte un échafaudage biocompatible et une quantité efficace de cellules précurseurs dermiques et épidermiques.
PCT/US2009/065202 2008-11-20 2009-11-19 Compositions et procédés de génération d’unités pilo-sébacées WO2010059862A2 (fr)

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KR101807727B1 (ko) 2017-10-23 2018-01-18 차의과학대학교 산학협력단 인간-유래의 체세포로부터 역분화 만능 줄기세포의 제조방법

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