SUPPORT FOR WOUND HEALING

专利摘要:
support for wound healing and method for promoting said healing. the present invention is directed to supports for wound healing co-grafted with a population of stem cells, in which the population of stem cells are abcb5 + stem cells. the supports are, for example, collagen glycosaminoglycan supports.
公开号:BR112015028193B1
申请号:R112015028193-1
申请日:2014-05-09
公开日:2020-10-06
发明作者:Markus H. Frank；Natasha Y. Frank；Dennis P. Orgill；George F. Murphy
申请人:Children's Medical Center Corporation；The Brigham And Women's Hospital, Inc.；Va Boston Healthcare System；
IPC主号:

专利说明:

RELATED PATENT APPLICATIONS
[001] This patent application claims priority under 35 USC § 119 (e) for US Provisional Patent Application No. Serial 61/822134, entitled "WOUND HEALING AND TISSUE ENGINEERING", filed on May 10, 2013, which this order is incorporated by reference in its entirety. GOVERNMENT SPONSORSHIP
[002] This invention was made with government support under NIH / NCI grant numbers 5R01CA113796. The government has certain rights in this invention. FIELD OF THE INVENTION
[003] The present invention is directed to methods and compositions of wound healing and tissue engineering, involving ABCB5 positive stem cells in collagen glycosaminoglycin supports. BACKGROUND OF THE INVENTION
[004] Regenerative medicine involves repair, regeneration, maintenance and replacement of tissues and organs using exogenous materials as supports. The supports can be seeded with cells, such as primary cells or stem cells and various factors to stimulate tissue growth. However, several challenges remain in the design of the appropriate material for regenerative medicine and tissue engineering.
[005] More than one million new chronic wounds develop in the United States each year with estimated treatment costs reaching billions of dollars. Wounds can be conceptualized as defects in the protective covering of an individual organ or organ system. Without this physiological barrier, the tissue normally protected by the cover is subject to loss of biological compartmentalization. When the tissue is no longer physiologically compartmentalized, it is subject to fluid loss, invasion by microorganisms, electrolyte imbalance and, in some cases, metabolic dysfunction. Fluids lost by non-compartmentalized tissue include, but are not limited to: blood, plasma, lymph, enteric contents, bile, cerebral spinal fluid, mucus. These fluid losses lead to desiccation of the underlying tissue and allow invasion by microorganisms, leading to potential infection and, in many cases, progressive tissue loss. For example, the inability to heal a chronic skin wound in the lower extremity can lead to amputation of a portion or the entire affected limb. There are several etiologies for such chronic skin wounds of the lower extremity, including mechanical wounds, burns, radiation, arterial insufficiency, venous stasis, chronic infection, neuropathy and systemic diseases such as diabetes. Current methods for improving wound healing enhance effective drainage, preventing infection, reducing inflammation and minimizing tissue and fluid loss.
[006] Chronic skin wounds impose significant health problems for patients with various diseases such as diabetes, burns, trauma such as trauma sustained by wartime, spinal cord injury and vascular insufficiency. Some of these patients are at risk of developing chronic wounds as a result of immobility and pressure ulcers as well as chronic ulcers that do not heal due to diabetes or peripheral vascular disease. The standard response to injury to human postnatal skin is driven by the need for rapid wound closure and is intended to result in the formation of a scar. Although scars are sufficient to restore the function of the skin barrier, they often impair other normal functions by replacing essential skin structures with connective tissue. In fetal life, a more protected environment in which skin development is bathed in sterile amniotic fluid, the human skin is fully capable of regeneration without scarring, that is, regenerative wound healing. The current understanding of the inherent plasticity of adult stem cells suggests that this phenomenon can be replicated in the postnatal period. SUMMARY OF INVENTION
[007] The present invention incorporates and is based at least in part on the discovery that stem cells that express ABCB5 show differentiation plasticity and even increase wound healing and / or tissue regeneration when used alone or in context of biodegradable supports.
[008] In some aspects, the invention is a support for wound healing comprising a support of collagen glycosaminoglycan co-grafted with a population of stem cells, in which at least 80% of the population of stem cells are ABCB5 + stem cells.
[009] In other aspects, the invention is a support for wound healing comprising a support of collagen glycosaminoglycan co-grafted with a population of stem cells, less than 50% of the cells in the composition are ABCB5 cells (-).
[0010] In still other aspects, the invention is a support for wound healing comprising a support of collagen glycosaminoglycan co-grafted with a population of ABCB5 + stem cells, in which the cell population includes less than 5% of keratinocytes and / or epidermal cells.
[0011] A support for wound healing comprises a support of collagen glycosaminoglycan co-grafted with a population of ABCB5 + eye stem cells, in which the cell population is free of non-ocular cells, is provided in other aspects of the invention.
[0012] A wound healing support comprises a collagen glycosaminoglycan support grafted with a population of ABCB5 + stem cells isolated from an individual's tissue, in which the ABCB5 + stem cells were separated from other cells in the individual using an antibody specific to ABCB5, is provided in other respects.
[0013] ABCB5 + stem cells can be ABCB5 + dermal mesenchymal stem cells. In some modalities, at least 85% or 90% of the stem cell population are ABCB5 + stem cells.
[0014] In some embodiments, the support is a porous matrix of cross-linked collagen and glycosaminoglycan. Collagen can be, for example, bovine tendon collagen. In some embodiments, the glycosaminoglycan is selected from the group consisting of chondroitin 6-sulfate, chondroitin 4-sulfate, heparin, heparin sulfate, keratin sulfate, dermatan sulfate and combinations thereof.
[0015] The support may include a semipermeable layer such as polysiloxane (silicone). In other modalities, the support is a mesh support. Optionally, the support can be formed for insertion into a fabric.
[0016] In some modalities, the support is the INTEGRA® Mesh Bilayer Healing Matrix.
[0017] The support can have a varied pore size. For example, the support may have a pore size of approximately 10 to 500 or approximately 50 to 350 or approximately 70 to 200 micrometers.
[0018] The support can include at least one bioactive molecule effective to increase wound healing. For example, the bioactive molecule may be a selected member of the group consisting of growth factors, anti-inflammatory agents, wound healing agents, anti-healing agents, antimicrobial agents, cell adhesion peptides, tissue generation modulating cells, nucleic acids, nucleic acid analogs, proteins, peptides, amino acids, ceramics, and combinations thereof.
[0019] In some embodiments, the support is adjusted as 25 cm2 (2 in. X 2 in.), 125 cm2 (4 in. X 5 in.), 250 cm2 (4 in. X 10 in.), Or 500 cm2 (8 in. X 10 in).
[0020] A method for promoting wound healing by placing a wound in contact with the wound healing support described in this application in order to promote healing is provided in other aspects of the invention. In some embodiments, contact comprises applying the composition to a bleeding site to control the bleeding.
[0021] In some modalities, the wound is a burn or a diabetic ulcer.
[0022] In some embodiments, a negative pressure wound therapy is used with the support.
[0023] In other modalities, the method may later involve maintaining the wound with a medically acceptable cover to treat the wound.
[0024] The wound can be selected from the group consisting of: partial and complete thickness wound, pressure ulcers, venous ulcers, diabetic ulcers, chronic vascular ulcers, tunneling / detaching wounds, surgical wounds, trauma wounds and wounds with drain.
[0025] The invention, in other aspects, is a method for tissue engineering, by sowing a collagen glycosaminoglycin support with ABCB5 + stem cells and maintaining the support under conditions such that the tissue is formed. In some embodiments, the tissue engineering method is a tissue regeneration method and the support is maintained under conditions such that the tissue is regenerated. In still other modalities, the tissue regeneration method is a method for treating aged skin.
[0026] A method for tissue engineering by seeding a biological tissue support with ABCB5 + stem cells and maintaining the support under conditions such that the tissue is formed, is provided in other aspects of the invention.
[0027] The biological tissue support can be, for example, an allograft or autograft, a xenogeneic tissue and / or decellularized tissue.
[0028] Other advantages and new attributes of the present invention will become evident from the following detailed description of various non-limiting modalities of the invention when considered together with the accompanying figures. In cases where the present specification and a document incorporated by reference include conflict and / or inconsistent disclosure, this specification will control.
[0029] Various methods are described in this application for administration to an individual of a composition for the treatment of a particular condition. It should be understood that in each such aspect of the invention, the invention specifically also includes the composition for use in the treatment of that particular condition, as well as use of the composition for the production of a medicament for the treatment of that particular condition.
[0030] This invention is not limited in its application to the details of the construction and the arrangement of components presented in the following description or illustrated in the drawings. The invention is capable of other modalities and can be practiced or carried out in various ways. In addition, the phraseology and terminology used in this application are for description purposes and should not be considered as a limitation. The use of "including", "comprising", or "having", "containing", "involving", and variations thereof in this application, is intended to encompass the items listed below and equivalents as well as additional items. BRIEF DESCRIPTION OF DRAWINGS
[0031] The accompanying drawings are not intended to be drawn to scale. In the drawings, each identical or nearly identical component that is illustrated in several figures is represented by a similar numeral. For the sake of clarity, not every component can be marked in each drawing. In the drawings:
[0032] Figure 1. Potential for multipotent differentiation of ABCB5 + cells. Immunofluorescence labeling (top three lines) of human skin ABCB5 + (left panel) or ABCB5- (right panel) cells for spectrin expression (myogenesis assay), CD31 (angiogenesis assay) and TUJ1 (neurogenesis assay), before and after differentiation. The cores are visualized with DAPI. Two bottom lines: Oil Red marking (adipogenesis assay) and Alizarin Red (osteogenesis assay) from ABCB5 + (left panel) or ABCB5- (right panel) of human skin cells before and after differentiation. The aggregated analysis of the pixel intensity of each marker (in myogenesis, angiogenesis and neurogenesis assays), or percentage of cells that mark positively (in adipogenesis and osteogenesis assays), in specimens in replicate (n = 3) are shown in bar diagrams on the right. *, P <0.05.
[0033] Figure 2. Schematic diagram of the murine ABCB5 gene locus and protein topology. The murine Abcb5 gene contains 28 exons and reaches 102 kb of genomic DNA at the 12qF2 locus. Encodes a 1255 AA protein with 11 transmembrane propellers and 5 extracellular loops. Exon 23 encodes AA 911-957, which forms an extracellular loop that contains the binding epitope of the anti-ABCB 3C2-1D12 antibody.
[0034] Figure 3. Analysis of mice with Abcbõ WT (upper panels) and Abcbõ KO (lower panels). H&E marking shows the thickness of the dermis with reduced subcutaneous fat and disorganized smooth muscle in KO animals (same magnification for WT and KO specimens). IHC and flow cytometry show complete loss of Abcbõ protein expression in Abcbõnull / null mice. Rhodamine-123 efflux studies have identified a cell population again with dye retention in Abcbõnull / null mice (R1 port, lower right panel), consistent with the loss of a distinctive Abcbõ function, the Rhodamine-123 efflux.
[0033] Figure 4A-4C. Regulation of stem cell quiescence by ABCBõ. Representative flow cytometric analyzes in two colors of murine skin cells from Abcbõ WT (B) and Abcbõ KO (C) mice labeled with BrdU in vivo and from unmarked Abcbõ WT controls (A). The cells are co-labeled with the anti-Brd11 antibody FITC and 7-AAD. Brdu-positive cells in the GO phase of the cell cycle are shown at port R1. BrdU-negative cells in S / G2 / M phases of the cell cycle are represented at the R2 port.
[0036] Figure 5A-5B. Differential expression of genes involved in cell cycle regulation between Abcbõ KO mice and Abcbõ WT mice. (A) A list of down-regulated genes in Abcbõ KO against Abcbõ WT mice as determined in real-time PCR analyzes. (B) The genes represented here are down-regulated in Abcbõ KO mice. The arrowed lines show known gene interactions. Gene relationships for canonical pathways such as p3 signaling, G1 / S control regulation, cyclins and cell cycle regulation, and calcium signaling pathways are detailed with lines without arrows. Genetic relationships and interactions are based on Via Ingenuity Analysis (Ingenuity, CA).
[0037] Figure 6A-6C. Comparative wound healing analyzes of Abcbõ KO and Abcbõ WT mice. (A) Digital photographs representative of day 0 and day 7 of complete thick skin wounds generated in Abcb5 WT (upper panel) and Abcbõ KO (lower panel) mice. (B) H&E marking representative of central wound cross sections, surrounding skin and underlying muscle tissue collected on experimental day 7 from Abcbõ WT (upper panel) and Abcbõ KO (lower panel) mice. (C) Quantitative analysis of wound closure (upper panel) and thickness of the inflammatory stroma (lower panel) of Abcb5 KO and Abcbõ WT wounds.
[0038] Figure 7A-7C. Comparative CD31 expression analysis in Abcbõ KO and Abcbõ WT mice. (A) and (B), H&E staining representative of CD31 of central wound cross sections, surrounding skin and underlying muscle tissue collected on experimental day 7 of Abcbõ WT (upper panel) and Abcbõ KO (lower panel) mice. (C) Quantitative analyzes of the CD31 + vascular layer thickness (upper panel) and cD31-avascular layer thickness (lower panel) in wounds of Abcbõ KO and Abcbõ WT.
[0039] Figure 8. Comparative analysis of vessel formation in Abcbõ KO and Abcbõ WT mice. CD31 labeling representing vascular layers of central cross sections of tissues collected on experimental day 7 of Abcbõ WT (left panel) and Abcbõ KO (right panel) mice.
[0040] Figure 9A-9B. Differential expression of genes involved in angiogenesis in Abcbõ KO against WT mouse wounds. (A) Levels of gene expression as determined by real-time PCR analysis. (B) Pro-angiogenic cytokines down-regulated in Abcbõ KO wounds are shown and marked. The arrows indicate the pro-angiogenic effect. An antiangiogenic transmembrane receptor, Bail, which is overexpressed in wounds of Abcbõ KO. The bar at the bottom indicates the antiangiogenic effect. Genetic relationships are based on Via Ingenuity Analysis (Ingenuity, CA).
[0041] Figure 10A-10D. Effect of ABCB5 + cells on wound healing in NSG mice. (A) Quantitative analyzes of the thickness of the inflammatory stroma of wounds inflicted in four experimental groups. (B) H&E staining representative of cross sections of wounds collected on experimental day 14. (C) Detection of human cells injected into INTEGRA® matrices by β2M specific for humans 14 days after transplantation. The black arrows point to clusters of human β2M + cells and individual human β2M + cells. (D) RT-PCR analyzes of cross sections of murine wounds for the expression of human-specific GAPDH and, β2-microglobulin and murine β-actin used as a load control.
[0042] Figure 11A-11C. Mouse human xenograft model showing the human skin graft established on the back of the 8-week post-graft mouse (A). Corresponding histopathology demonstrates the anastomosis of human and murine skin is shown in (B). The human xenograft wound (C) allows immunohistochemical detection of specific dermal cells and elements of the extracellular matrix at time points 0, 2, 4, and 7 days post-wound (top to bottom).
[0043] Figure 12. Role of ABCB5 in human regenerative wound healing. Comparative studies that examine the immunohistochemical profiles of consequent healing responses for scar formation and regeneration induced by INTEGRA® support (scar v. Support) can be refined (note the remarkable realignment of actin-expressing myofibroblasts and marked expression increased number of ABCB5 + dermal cells in the wound carrying the support). DETAILED DESCRIPTION
[0044] The present invention is based in part on the discovery that collagen glycosaminoglycan supports seeded with ABCB5 positive stem cells demonstrate increased wound healing and tissue engineering properties. It has been shown that the constructs of the invention have unique regenerative activity that leads to tissue synthesis. Enhanced tissue synthesis is useful in tissue repair and generation and wound repair and healing.
[0045] Useful cells according to the invention are ABCB5 positive stem cells. ABCB5 is an important new marker for the isolation of multipotent stem cell populations from normal human tissue. "ABCB5 (+) stem cells", as used in this application, refers to cells that have the ability to self-renew and differentiate into old cells from multiple adult cell lines. These cells are characterized by the expression of ABCB5 on the cell surface. In some embodiments of the invention, ABCB5 (+) stem cells are dermal or ocular stem cells.
[0046] "ABCB5 positive dermal mesenchymal stem cells", as used in this application, refers to skin cells that have the ability to self-renew and differentiate into old cells from multiple adult cell lines such as bone, fat and cartilage. These cells are characterized by the expression of ABCB5 on the cell surface. In culture, mesenchymal stem cells can be guided to differentiate into bone, fat, cartilage or muscle cells using specific means. (Hirschi KK e, Goodell MA. Gene Ther. 2002; 9: 648-652. Pittenger MF, et al., Science. 1999; 284: 143-147. Schwartz RE, et al., J Clin Invest.2002; 109 : 1291-1302. Hirschi Ke Goodell M. Differentiation. 2001; 68: 186-192.)
[0047] ABCB5 positive dermal mesenchymal stem cells can be obtained from the skin. The skin can be derived from any individual who has skin, but in some modalities it is preferably human skin. The skin can be derived from an individual of any age but in some modalities it is preferably adult skin, rather than adolescent or child skin.
[0048] In other embodiments of the invention, ABCB5 (+) stem cells are retinal stem cells. ABCB5 (+) stem cells can be obtained (for example, isolated or derived from) from the basal limbic epithelium of the eye or from the retinal pigment epithelium (RPE). In some embodiments, ABCB5 (+) stem cells are obtained from the human eye. Other types of ABCB5 (+) stem cells, such as, for example, those obtained from the central cornea can be used in various aspects and modalities of the invention.
[0049] ABCB5 (+) stem cells can be isolated. An "isolated ABCB5 (+) stem cell", as used in this application, refers to a cell that was removed from an organism in which it was originally found, or a descendant of such a cell. An isolated cell also refers to a cell that is placed in conditions beyond the natural environment. Such a cell can then be introduced into a second organism or reintroduced into the organism from which it (or the cell or population of cells from which it descended) was isolated. It is still considered that such a cell, once manipulated according to the methods of the invention, is an isolated cell. The term "isolated" does not prevent the subsequent use of the cell after it in combinations or mixtures with other cells or in an in vivo environment.
[0050] ABCB5 (+) stem cells can be obtained from an individual by isolating a tissue sample, including skin cells, such as dermal cells and eye cells from the basal limbic epithelium or RPE, and then purifying the ABCB5 stem cells (+). It will be apparent to those skilled in the art that a sample can be enriched for ABCB5 + stem cells in a variety of ways. For example, stem cells can be selected to use antibodies or other binding molecules that bind to molecules on the ABCB5 cell surface in cells. Stem cells can be obtained directly from a donor or recovered from cryopreservative storage. Stem cells can, for example, be isolated using antibodies against ABCB5 and maintained in culture using standard methodology or frozen, for example, in liquid nitrogen, for later use.
[0051] Populations of specifically pure ABCB5 + dermal cells with mesenchymal stem cell molecular phenotype can be isolated from surgical specimens of healthy human skin using an established, sensitive and specific monoclonal antibody (mAb), for example. The isolated ABCB5 + dermal stem cells have a capacity for multipotent differentiation such that they differ in cell lines from the three germ layers, that is, ecto-derma, mesoderm and endoderm.
The present invention contemplates any method suitable for employing ABCB5 binding molecules such as, for example, monoclonal antibodies, polyclonal antibodies, human antibodies, chimeric antibodies, humanized antibodies, single chain antibodies, F (ab ') 2, Fab, Fd, Fv or Fv single chain fragments to separate ABCB5 (+) stem cells from a varied population of cells. Consequently, the methods include a method of producing an ABCB5 (+) stem cell population that comprises the steps of providing a cell suspension of cells; contacting the cell suspension with a monoclonal antibody or combination of monoclonal antibodies, which recognize an epitope, including ABCB5, in ABCB5 (+) cells; and separating and recovering cells bound by monoclonal antibodies from the cell suspension. Monoclonal antibodies can be linked to a solid phase and used to capture ABCB5 + stem cells. The bound cells can then be separated from the solid phase by known methods depending on the nature of the antibody and the solid phase.
[0053] "Monoclonal antibody", as used in this application, refers to an antibody obtained from a single clonal population of immunoglobulins that bind to the same epitope as an antigen. Monoclonal-based systems suitable for preparing cell populations of the invention include magnetic beads / column of paramagnetic particles that use antibodies for positive or negative selection; affinity-based separation of biotin or streptavidin; and high-speed flow cytometric sorting of immunofluorescent labeled LSCs and mixed in a suspension of other cells. Accordingly, the methods of the present invention include isolating a population of cells and augmenting using monoclonal antibodies raised against the ABCB5 surface antigen (for example, monoclonal antibodies that selectively bind to ABCB5). In some examples, commercially available antibodies or antibody fragments that selectively bind to ABCB5 can be used in the methods described in this application. Such antibodies are considered to selectively bind to ABCB5 if they bind or are capable of binding to ABCB5 with a greater affinity than the affinity with which monoclonal antibodies can bind to other antigens (that is, except ABCB5 antigens). Such binding can be measured or determined by standard protein-protein interaction assays (for example, antibody-antigen or ligand-receptor assays) such as, for example, competitive assays, saturation assays or standard immunoassays including, without limitation, immunoab assays - enzyme-linked sorbents, radioimmunoassays and radioimmunofilter binding assays.
[0054] ABCB5 (+) stem cells can be prepared as substantially pure preparations. The term "substantially pure", as used in this application, refers to a preparation that is substantially free of ABCB5 (+) cells in addition to stem cells. For example, a substantially pure ABCB5 (+) stem cell preparation may constitute a preparation in which at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90% at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% of the total of the cells present in a preparation are ABCB5 (+) stem cells.
[0055] The compositions of the invention comprise a substrate such as, for example, a biocompatible material that promotes wound healing, including biodegradable supports. ABCB5 (+) stem cells can be added to the substrate or support to form, for example, tissue or tissue grafts for transplantation. The support is a highly porous lattice comprised of collagen and glycosaminoglycan, that is, a matrix or support of collagen glycosaminoglycan. Examples of collagen glycosaminoglycan supports include those listed in U.S. Pat. U.S. Nos. 4,060,081, 4,280,954 and 4,505,266. Other materials useful in collagen glycosaminoglycan supports include, but are not limited to, chondroitin 6-sulfate, chondroitin 4-sulfate, heparin, heparin sulfate, keratin sulfate, dermatan sulfate, chitin and chitosan. Collagen glyco-saminoglycan supports serve as a support or structure system of structures in which blood vessels and surrounding tissue cells migrate from within a tissue cavity, a process referred to as "infiltration". The infiltration is responsible for creating a new tissue, which replaces the support as it biodegrades.
[0056] In some modalities, the support is INTEGRA®. INTEGRA® is the FDA approved acellular dermal skin substitute comprising the extracellular matrix (collagen and GAG). It has been used to treat large tissue defects or wounds if they do not heal like venous leg ulcers.
[0057] "Compositions" in this application may refer to isolated cell preparations or supports, including fabric supports and artificial supports. The compositions of the invention, in some examples, are enriched by isolated ABCB5 (+) stem cells. A composition is considered to be enriched by isolated ABCB5 (+) stem cells if ABCB5 (+) stem cells are the predominant cell subtype present in the preparation. For example, an ABCB5 (+) stem cell enriched composition is a composition in which at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% of the cells in the composition are ABCB5 (+) stem cells. In some embodiments, a composition enriched with isolated ABCB5 (+) stem cells is one in which less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2% or less than 1% of the cells in the composition are ABCB5 (-) cells. In some embodiments, the cells in a composition are only dermal cells. For example, the support is seeded with cells such that all cells are ABCB5 + dermal cells. That is, in some embodiments, a composition may not contain non-dermal cells. In some embodiments, the cells do not include keratinocytes and / or epidermal cells. In some modalities, the cells in a composition are just eye cells. That is, in some embodiments, a composition may not contain non-ocular cells. In some embodiments, a composition may not contain ocular ABCB5 (-) cells.
[0058] The supports of the present invention can be formatted in a variety of ways, as a single sheet, or as a laminated sheet containing multiple layers or sheets of collagen. In certain embodiments, the supports comprise 2 to 15 sheets. Such sheets can be held together by seams or sutures.
[0059] In a particular embodiment, the polymer further comprises a bioactive molecule, for example, a small molecule or a peptide in addition to stem cells. The bioactive molecule can be non-covalently incorporated into the polymer, for example, as a suspension, encapsulated as particles, microparticles or colloids, or as a mixture thereof. The bioactive molecule can also be covalently incorporated into the polymer, using any suitable chemistry for binding the bioactive molecule to the polymer. The bioactive molecule can be any therapeutically desirable molecule, such as a growth factor, an antimicrobial, an analgesic, a hematostatic, a pro-angiogenic agent or an antiangiogenic agent. In exemplary embodiments, the polymer comprises one or more of FGF2, NGF, doxycycline, amoxicillin and poly-L-lysine.
[0060] In another particular modality, the supports have a width of at least 10 cm. For example, the support can be at least 10 cm wide and at least 10 cm long. Consequently, certain supports can have a surface area of more than 100 cm2, for example, 400 cm2. The supports of the invention can have a biaxial force of at least 80 N or more.
[0061] The tissue supports of the invention can be used in multiple applications including, but not limited to, covering a tissue deficit or wound, reinforcing the tissue as soft tissue, and organ / tissue generation or regeneration. Consequently, in another aspect, the invention features a method for inducing the repair of a damaged tissue, comprising bringing the damaged tissue into contact with a support of the invention. The invention further features a method for stimulating soft tissue regeneration, comprising bringing the soft tissue into contact with a support of the invention. When a support is placed in contact with a tissue, the support can increase the proliferation of cells located near the support. In addition, the support can promote vascularity within a tissue to which it adheres. Consequently, in another aspect, the invention provides a method of stimulating cell proliferation in a tissue, comprising bringing the tissue into contact with a support such that cell proliferation is stimulated. The invention further provides a method of inducing vascularization of a tissue, comprising bringing the tissue into contact with a support such that vascularization occurs within the tissue.
[0062] The support can be formed to fill a defect in the fabric. In most cases, this can be achieved by cutting the polymer fibers with scissors or a knife; alternatively, the support can be released from a polymer solution formed by heating or dissolving in a volatile solvent.
[0063] Mesenchymal stem cells are seeded on the support by applying a cell suspension to the support. This can be accomplished by soaking the support in a cell culture vessel, or injection or other direct application of the cells to the support.
[0064] The support sown with cells is implanted at the defect site using standard surgical techniques. The support can be seeded and grown in vitro before implantation, seeded and immediately implanted, or implanted and then seeded with cells. In one embodiment, cells are seeded to and in support and cultured in vitro between approximately sixteen hours and two weeks, although it may be a longer period. Cell density at the time of sowing or implantation will vary under these circumstances. For example, cell density can be approximately 25,000 cells / mm3. The specialist will recognize the appropriate cell density.
[0065] As used in this application, an individual can be a mammal such as, for example, a human, non-human primate, cow, horse, pig, sheep, goat, dog, cat or rodent. Human ABCB5 (+) stem cells and human individuals are particularly important modalities.
[0066] In some embodiments, isolated ABCB5 (+) stem cells (for example, as a composition in the form of an ABCB5 (+) stem cell graft or as a preparation of cells delivered to an implanted graft) can be administered to an individual more than once. Thus, in some modalities, an individual can be administered multiple doses or grafts (for example, 2, 3, 4 or more) of isolated ABCB5 (+) stem cells over the course of several weeks, months or years. In some modalities, stem cells are administered again 3 months, 6 months, 9 months, 12 months, 18 months, 21 months or 24 months after the first application. The number of applications and the frequency of application may depend, for example, on the degree of cell regeneration achieved after the first administration / transplantation of stem cells. The number and frequency of stem cell applications can be determined by a medical professional (for example, surgeon, physician).
[0067] The compositions of the invention (support seeded with ABCXB5 + stem cells) are useful in wound healing. Most wounds on the skin and other organ systems are characterized by a loss of cells and connective tissue matrix from the protective outer layer as well as the underlying layers and tissues. In case of skin wounds, the epidermis is the outer layer that is lost. The epidermis covers the dermis as well as deeper structures like fat, muscle and bone. Closing large wounds in the skin and other organ systems typically requires the production of billions of cells, nutrition by a vascular network and the mechanical strength of proteins and glycosaminoglycans present in a nascent extracellular matrix (ECM).
[0068] The term "wound", for the purposes of this application, broadly refers to an injury to an organ or system of organs. In the case of the skin, the lesion may be in the epidermis, dermis and / or subcutaneous tissue. Skin wounds can be classified into one of four degrees depending on the depth of the wound: i) Grade I: wounds limited to the epithelium; ii) Grade II: wounds that extend on the dermis; iii) Grade III: wounds that extend into the subcutaneous tissue; and iv) Grade IV (or full thickness wounds): wounds to which the bones are exposed (for example, a point of bone pressure such as the greater trochanter or sacrum). The term "partial thickness wound" refers to wounds that encompass Grades I to III; examples of partial thickness wounds include burn wounds, pressure sores, venous stasis ulcers and diabetic ulcers. The term "deep wound" includes both Grade III and Grade IV wounds. The methods of the invention are useful for treating all grades of wounds, including chronic and acute wounds. The term "chronic wound" refers to a wound that has not healed within 30 days.
[0069] The term "promoting wound healing", for the purposes of this application, refers to the ability to rebuild the normal physiological barrier of an organ or organ system. In the case of skin wounds, promoting wound healing may include the induction of granulation tissue formation, and / or the induction of wound contraction, and / or the induction of revascularization and / or the induction of epithelialization ( that is, the generation of new cells in the epithelium). In some embodiments, ABCB5 + cells may function at least in part by secreting mediators, such as, for example, VEGF.
[0070] The types of wounds to be treated by the methods of the invention include various types of wounds including, but not limited to: surgical wounds; traumatic wounds; radiation injury wounds; wounds of toxic epidermal necrolysis; infectious wounds; neoplastic wounds; full thickness wounds; partial thickness wounds; and burn wounds, as well as wounds that result from the various types of ulcers, such as skin ulcers, corneal ulcers, obstructive arterial ulcers, pressure-induced and diabetic decubitus ulcers, burn ulcers, injury ulcers, radiation ulcers , drug-induced ulcers, post-operative ulcers, inflammatory ulcers, ulcers of the gastrointestinal tract, simple ulcers and other types of angiopathic ulcers and chronic (intractable) ulcers.
[0071] The methods of various embodiments of the invention can be particularly useful in the treatment of complex wounds or wounds of difficult healing. Many factors can adversely affect the wound healing process, including infection, irradiated tissue, systemic disease, medications, patient age, patient health, and the individual's nutritional status. In addition, any process that prevents peripheral blood circulation, such as arteriosclerosis, prolonged pressure, varicose vein disease and venous stasis, can adversely affect the delivery of oxygen, nutrients, chemical signals, and appropriate cell types to mediate healing in a injured individual, will impair wound healing. Factors that inhibit wound healing include wound desiccation, medication such as chemotherapy or steroids, and poor patient health and / or nutrition. Certain partial and complete thickness lesions, such as burns, skin grafts, and various types of ulcers, resist repair and produce significant pain and discomfort to the individual.
[0072] The general physical condition of the patient is also important in wound healing. As age increases, the ability to repair damaged tissue decreases as the skin becomes thinner and the number of fibroblasts and the total amount of skin collagen decreases. Disease states such as alcoholism, anemia, diabetes, malnutrition, shock and uremia lead to impaired delivery of oxygen and nutrients to the wound site, thereby inhibiting the healing process. In addition, diseases that lead to monocytopenia can significantly impair wound healing.
[0073] Medications used to treat disorders can produce impaired wound healing. Chemotherapy, used to eliminate cells that divide into cancer patients, also suppresses such a patient's ability to heal wounds, which also depends on new cell growth. Steroids negatively impact the three stages of wound repair, inhibiting the initial inflammatory response, reducing the production of new epithelium and vascular tissue, and weakening the collagen matrix in the scar tissue.
[0074] Bacterial wound infection is a common local cause of prolonged wound healing. Human skin is typically colonized by several microorganisms, including Candida albicans, Staphylococcus epidermidis, Staphylococcus aureus and some strains of Streptococcus, so any wound that exposes tissues underlying the environment is at least infected with the resident microbial flora. Wounds that are well cared for and in highly vascularized tissue resist infection, while those in ischemic tissue are much more susceptible to infection.
[0075] In some modalities, the individual may have a skin wound. In other embodiments, the individual may have an eye condition such as an eye wound (for example, dead, damaged or infected eye cells), for example, in the corneal epithelium. Thus, the corneal epithelium can be injured in an individual who has an eye condition according to the invention.
[0076] In some embodiments, the support of the invention can be combined with a device to exert pressure on a wound. The cells within the wound can be subjected to controlled tension using devices that mechanically induce tension or compression in a constant or time-dependent manner as needed. To apply localized controlled forces to a wound surface, a device that has several microchannels fluidly connected to microstructures, such as microchambers, for example, within a matrix that can be positioned on a wound surface can be used. The vacuum pressure (or positive pressure) applied to each microchamber is controlled via microchannels. The term "vacuum pressure", for the purposes of this application, refers to a pressure in a chamber or material of interest that is less in magnitude than that of a reference chamber, material, fabric or atmosphere. The term "positive pressure", for the purposes of this application, refers to a pressure in a chamber or the material of interest that is higher in magnitude than that of a reference chamber, material, fabric or atmosphere. The term "pressure", for the purposes of this application, is intended to encompass both vacuum pressure and positive pressure. The support of the invention can be applied to the wound before, after, or intermittently with a device for applying pressure.
[0077] Wound healing involves the formation of a fibrin clot, the recruitment of inflammatory cells, re-epithelialization, and matrix formation and remodeling. Immediately after tissue damage, rupture of the blood vessel leads to blood leakage and accompanying platelet aggregation and blood clotting that results in fibrin clot formation. Activated platelets captured within the fibrin clot degranulate and release a variety of cytokines and growth hormones. These cytokines and growth hormones help to recruit inflammatory cells to the lesion site, stimulate angiogenesis and initiate tissue movements associated with re-epithelialization and contraction of connective tissue.
[0078] Neutrophils and monocytes are recruited to the lesion site by various chemotactic signals including growth factors and cytokines released by degranulation platelets, formyl methionyl peptides cleaved from bacterial proteins and by-products of fibrin proteolysis and other matrix proteins. Neutrophil infiltration ceases after a few days, but macrophages continue to accumulate by the continuous recruitment of monocytes to the wound site. Activated macrophages release growth factors and cytokines which thereby amplify the early signs of platelets in degranulation. Exogenous factors can be applied to the wound to aid in these processes.
[0079] Thus, the modalities of the invention also include methods that involve the inclusion of soluble factors with ABCB5 + cells. Following the placement of the device on the wound, the soluble factors added to the device (e.g., growth factors such as epidermal growth factor, cytokines, PGDF, insulin-like growth factor, TGF-beta, keratinocyte growth factor cytokine, TNF, chemokines, chemotactic peptides, tissue inhibitors of metalloproteinases, etc.) pass into the tissue.
[0080] It has been observed that several recombinant growth factors can accelerate the wound healing process, both in acute and chronic wounds, in animal models. These recombinant derived factors include Platelet Derived Growth Factor (PDGF), Fibroblast Growth Factor (FGF), Epidermal Growth Factor (EGF), and Growth Transformation Factors α and β (TGF-α and TGF-β) . In addition, other recombinant growth factors, including insulin, Growth Factors similar to Insulin I and II (IGF-I and IGF-II, respectively), Interferons (IFNs), Interleukins (ILs), KGF (Que- ratinocyte), Macrophage Colony Stimulation Factor (M-CSF), Platelet-derived Endothelial Cell Growth Factor (PD-ECGF), and Stem Cell Factor (SCF), may promote activation, proliferation and / or stimulation of cell types involved in the wound healing process.
[0081] Soluble factors can be proteins or can be expressed in cells. The protein, peptide or polypeptide refers to a polymer of amino acids, and these terms are used interchangeably. The polymer can include natural or unnatural amino acids. The protein or polypeptide can be produced in vitro or in vivo by natural, recombinant, synthetic or other means. The protein or polypeptide may have post-translational modifications or may have been chemically modified to include phosphorylation, glycosylation, famesylation, acetylation, methylation, thiol oxidation, etc.
[0082] Another use of the compositions of the invention is in tissue regeneration. Wound healing can be achieved through tissue repair or through tissue regeneration. In comparison with the repair, which usually results in the formation of a scar, tissue regeneration provides complete morphological and functional restoration of normal structures. Spontaneous tissue regeneration does not happen in postnatal life; however, at least it can be partially aided by exogenous biological matrices, such as supports, clinically known as INTEGRA® (Integra LifeSciences, Plainsboro, NJ), which have been approved by the United States Food and Drug Administration for use in patients massively burned and for the treatment of reconstructive defects and chronic wounds. The regenerated skin is mechanically competent, fully vascularized, and sensitive to touch and heat or cold6, but has no critical skin appendages, for example, hair follicles and sweat glands5.
[0083] Transplantation of grafts containing ABCB5-positive stem cells, but not ABCB5-deficient dermal grafts, will further increase the regenerative wound healing induced by INTEGRA® and potentially increase the formation of skin appendages due to increased local availability of regenerative multipotent stem cell populations associated with the response. Hereby it is expected that significant improvements in the wound healing response will be accompanied by simultaneous regeneration of the dermis and epidermis and reduced scarring.
[0084] In this aspect of the invention, supports seeded with ABCB5 positive cells are used to generate the tissue by inducing differentiation. Isolated and purified mesenchymal stem cells can be grown in an undifferentiated state through mitotic expansion in a specific medium. These cells can then be collected and activated to differentiate into bone, cartilage and various other types of connective tissue by several factors, including mechanical, cellular and biochemical stimuli. Human mesenchymal stem cells have the potential to differentiate into cells such as osteoblasts and chondrocytes, which produce a wide variety of mesenchymal tissue cells, as well as tension, ligament and dermis, and this potential is conserved after isolation and for various population expansions in the culture. Thus, being able to isolate, purify, multiply enormously, and then activate mesenchymal stem cells to differentiate into the specific types of desired mesenchymal cells, such as skeletal and connective tissues such as bone, cartilage, tendon, ligament, muscle and animal fat, a process exists to treat skeletal and other connective tissue disorders. The term connective tissue is used in this application to include tissues in the body that support specialized elements, and includes bone, cartilage, ligament, tendon, stroma, muscle and adipose tissue.
[0085] In another aspect, the present invention relates to a method for repairing connective tissue damage. The method comprises the steps of applying the supports to an area of connective tissue damage under suitable conditions to differentiate stem cells in the type of connective tissue needed for repair.
[0086] The term "connective tissue defects" refers to defects that include any damage or irregularity compared to normal connective tissue that may occur due to the wound, disease, age, birth defect, surgical intervention, etc. Connective tissue defects also refer to undamaged areas where bone formation is only desired, for example, for cosmetic enhancement.
[0087] The supports are also useful in the treatment of liver disease. Liver disease includes disease such as hepatitis that results in damage to liver tissue. More generally, the supports of the present invention can be used for the treatment of liver diseases, disorders or conditions including but not limited to: alcoholic liver disease, hepatitis (A, B, C, D, etc.) focal liver damage, hepatocellular carcinoma primary, large cystic lesions of the liver, focal nodular hyperplasia, granulomatous liver disease, liver granulomas, hemochromatosis such as hereditary hemochromatosis, iron overload syndromes, acute fatty liver, pregnancy hyperemesis, intercurrent liver disease during pregnancy, intra cholestasis - liver, liver failure, fulminant liver failure, asymptomatic jaundice or hyperbilirubinemia, hepatocyte injury, Crigler-Najjar syndrome, Wilson's disease, alpha-1-antitrypsin deficiency, Gilbert's syndrome, hyperbilirubinemia, non-alcoholic steatohepatitis , porphyrias, non-cirrhotic portal hypertension, portal fibrosis, schistosomiasis, primary biliary cirrhosis, Budd-Chiari, hepatic veno-occlusive disease after bone marrow transplantation, etc.
[0088] In some embodiments, the invention is directed to the treatment of a neurogenerative disease, with the supports of the invention. In some cases, the invention contemplates the treatment of individuals who have neurogenerative disease or damage to nerve cells that can lead to neurodegeneration. Neuronal cells are predominantly categorized based on their local / regional synaptic connections (for example, local circuit interneurons versus long-term projection neurons) and receptor sets, and associated with secondary messenger systems. Neuronal cells include both neurons in the central nervous system (CNS) and neurons in the peripheral nervous system (PNS). There are many different neuronal cell types. Examples include, but are not limited to, sensory and sympathetic neurons, cholinergic neurons, dorsal root ganglion neurons, proprioceptive neurons (in the trigeminal mesencephalic nucleus), ciliary ganglion neurons (in the parasympathetic nervous system), etc. One of ordinary skill in the art will be able to easily identify neuronal cells and distinguish them from non-neuronal cells such as glial cells, typically using cellular and morphological characteristics, expression of specific cellular markers, secretion of certain molecules, etc. "Neurogenerative disorder" or "neurogenerative disease" is defined in this application as a disorder in which the progressive loss of neurons occurs in the peripheral or central nervous system. These disorders include injury-related neuronal damage such as spinal cord injury and head injury.
[0089] Most chronic neurodegenerative diseases are typified at the beginning during middle adulthood and lead to rapid degeneration of specific subsets of neurons within the neural system, ultimately resulting in premature death. Compositions comprising dermal mesenchymal stem cells can be administered to an individual to treat neurogenerative disease alone or in combination with the administration of other therapeutic compounds for the treatment or prevention of these disorders or diseases.
[0090] The usefulness of adult stem cells in the treatment of neurodegenerative disease has been described. It has been shown that mesenchymal stem cells can change into neuron-like cells in mice that have experienced strokes. Journal of Cell Transplantation Vol. 12, pp. 201-213, 2003. Additionally, stem cells derived from bone marrow developed in neural cells keep the promise of treating patients with Parkinson's disease, amyotrophic lateral sclerosis (ALS) and spinal cord injuries.
[0091] The methods of the invention are also useful in the treatment of disorders associated with kidney disease. Mesenchymal stem cells previously injected into the kidneys have been shown to result in an almost immediate improvement in kidney function and cell renewal. Resnick, Mayer, Stem Cells Brings Fast Direct Improvement, Without Differentiation, in Acute Renal Failure, EurekAlert !, August 15, 2005. In this way, the supports of the invention can be administered to an individual who has kidney disease alone or in combination with other therapy or procedures, such as dialysis, to improve kidney function and cell renewal.
[0092] Other diseases that can be treated according to the methods of the invention include diseases of the cornea and lung. Therapies based on the administration of mesenchymal stem cells in these tissues have shown positive results. For example, human mesenchymal stem cells were used to reconstruct damaged corneas. Ma Y et al, Stem Cells, 18 August 2005. Additionally, stem cells derived from bone marrow were found to be important for lung repair and protection against lung injury. Rojas, Mauricio, et al., American Journal of Respiratory Cell and Molecular Biology, Vol. 33, pp. 145-152, May 12, 2005. In this way, the dermal mesenchymal stem cells of the invention can also be used to repair lung tissue or corneal tissue.
[0093] ABCB5 (+) stem cells can be autologous for the individual (obtained from the same individual) or non-autologous as cells that are allogeneic or syngeneic for the individual. Alternatively, ABCB5 (+) stem cells can be obtained from a source that is xenogeneic for the individual.
[0094] Allogeneic refers to cells that are genetically different although belonging to or obtained from the same species as the individual. Thus, an allogeneic human ABCB5 (+) stem cell is a stem cell obtained from a human being in addition to the desired stem cell container. Singeneic refers to cells that are genetically identical or closely related and immunologically compatible with the individual (that is, from individuals or tissues that have identical genotypes). Xenogeneic refers to cells derived from or obtained from an organism of a species other than the individual.
[0095] ABCB5 (+) stem cells, according to the invention, can be expanded ex-vivo or in vitro before application to the support or in vivo after administration. Thus, in some instances, the expression of ABCB5 provides a basis for identification, isolation, cloning, propagation and expansion of ABCB5 (+) stem cells in vitro. Any suitable method of employing agents, for example, isolated peptides, for example , antibodies, which bind to ABCB5 to separate ABCB5 (+) stem cells from other cells, can be used. Isolated ABCB5 (+) stem cells can be maintained in an appropriate culture environment using, for example, a combination of media, supplements and reagents. Optionally, feed cell populations or conditioned media obtained from feed cell populations can be used to expand ABCB5 (+) stem cell populations.
[0096] Adhesion, binding and matrix factors that can be used for the expansion of stem cells according to the invention include, without limitation, E-cadherin, collagen, fibronectin, superfibronectin, heparin sulfate proteoglycan, ICAM- I, laminin, osteopontin, proteoglycan, E-selectin, L-selectin, VCAM and vitronectin.
[0097] Bioactive and supplements that can be used for the expansion of stem cells according to the invention include, without limitation, enzymes (eg, cathepsin G, Flt-3 / Fc), proteins and peptides (eg, activin A, albumin, angiogenin, angiopoietin, BAX inhibiting peptide, heregulin beta-1, SMAC / Diablo), vitamins, hormones and various other substances (eg L-ascorbic acid, dexamethasone, EGF, EGF receptor, embryonic fluid (bovine ), flt3-ligand, progesterone, retinoic acid, retinyl acetate, thrombopoietin and TPO), antibodies, chemokines, cytokines, growth factors and receptors.
[0098] Culture reagents that can be used for stem cell expansion according to the invention include, without limitation, antibiotics (eg, cycloheximide, etoposide, gentamicin, mitomycin, penicillin-streptomycin), classic media (eg example, Claycomb Medium, Dulbecco Modified Eagle Medium, Iscove Modified Dulbecco Medium, Minimal Essential Medium), DMSO-cell freezing medium, Claycomb Medium without L-glutamine, Stemline® Medium (Sigma-Aldrich, USA).
[0099] The compositions of the present invention may comprise stem cells or isolated preparation of stem cells, the stem cells characterized by the expression of ABCB5 on their cell surface grafted with a glycosaminoglycan support. A composition can comprise a preparation enriched with isolated ABCB5 (+) stem cells, or can comprise a substantially pure population of ABCB5 (+) stem cells. The compositions are intended to encompass the supports discussed in this application.
[00100] The compositions, in some modalities, may comprise additional bioactive and supplements to promote cell regeneration and differentiation. Such bioactive and supplements that can be used in accordance with the invention are described above and include, without limitation, various enzymes, proteins and peptides, vitamins, antibodies, chemokines, cytokines, growth factors and receptors. In some embodiments, the compositions may comprise an immunosuppressant and / or an antivasculogenesis agent. For example, in some embodiments, a composition may comprise cyclosporine (for example, CyA), which can be used to prevent and / or treat graft rejections. In some embodiments, the compositions may comprise bevacizumab (for example, AVASTIN®). The use of antivasculogenesis agent can be used, in some instances, to prevent blood vessel formation, which often occurs after transplantation and can lead to graft rejection. In some embodiments, an immunosuppressant and / or an anti-cancer agent is not administered as a component of a composition or support, instead it is administered independently before or following the administration of ABCB5 (+) stem cells.
[00101] ABCB5 + cells can be engineered genetically or recombinantly. Recombinant can refer to organisms, cells, nucleic acids and proteins. Recombinant cells and organisms are cells and organisms that contain recombinant DNA. Recombinant DNA refers to a sequence of nucleic acids that is not normally found in nature. This term usually refers to two or more pieces of DNA linked together to form an unnatural product. Recombinant protein is protein produced from recombinant DNA (that is, a nucleic acid that differs from what occurs in nature). In the production of a recombinant protein, the regulatory sequences of the gene encoding the protein are usually different than those that occur in the natural gene. The gene may also have been placed in an organism that normally does not have the gene in order to produce that protein in the desired organism.
[00102] The insertion of desired genes or other nucleic acid constructs into cells seeded for support can be carried out using genetic engineering techniques and regular recombinants, for example, as described in Ausubel et al., Eds., 1989, Current Protocols in Molecular Biology, Green Publishing Associates, Inc. and John Wiley & Sons, Inc., New York.
[00103] Dermal mesenchymal stem cells can be modified to express proteins that are also useful in therapeutic indications, as described in more detail in this application. For example, cells may include a nucleic acid that produces at least one bioactive factor that further induces or accelerates the differentiation of mesenchymal stem cells into a differentiated strain and / or cells may include a nucleic acid that produces a secreted mediator . In the example where the bone is being formed, the bioactive factor may be a member of the TGF-beta superfamily that comprises several tissue growth factors, particularly bone morphogenic proteins, such as at least one selected from the group consisting of BMP-2, BMP -3, BMP-4, BMP-6 and BMP-7. In other examples, the secreted mediator can be VEGF.
[00104] Various techniques can be employed to introduce nucleic acids into cells. Such techniques include transfection of CaPO4-precipitated nucleic acids, transfection of nucleic acids associated with DEAE, transfection with a retrovirus including the nucleic acid of interest, liposome-mediated transfection, and the like. For certain uses, it is preferable to target the nucleic acid to particular cells. In such examples, a vehicle used to deliver a nucleic acid according to the invention into a cell (for example, a retrovirus or other virus; a liposome) can have a targeting molecule attached to it. For example, a molecule such as an antibody specific for a surface membrane protein in the target cell or a receptor ligand in the target cell can be linked to or incorporated into the nucleic acid delivery vehicle. For example, where liposomes are employed to deliver the nucleic acids of the invention, proteins that bind to a surface membrane protein associated with endocytosis can be incorporated into the liposome formulation for targeting and / or facilitating entry.
[00105] A method of introducing exogenous genetic material into dermal mesenchymal stem cells is by transducing the cells using replication-deficient retroviruses. The replication-deficient retroviruses are capable of directing the synthesis of all virion proteins, but are unable to make particles infectious. Consequently, these genetically altered retroviral vectors are of general use for highly efficient transduction of genes in cultured cells. Retroviruses have been used extensively to transfer genetic material to cells. Standard protocols for producing replication-deficient retroviruses (including steps for incorporating exogenous genetic material into a plasmid, transfection of a plasmid packaging cell line, production of recombinant retroviruses by the packaging cell line, the collection of viral particles of tissue culture media and infection of target cells with viral particles) are provided in the art.
[00106] The main advantage of using retroviruses is that viruses efficiently insert a single copy of the gene that encodes the therapeutic agent into the host cell genome, thereby allowing exogenous genetic material to be transmitted to the cell's progeny when it divides. In addition, it has been reported that gene promoter sequences in the LTR region increase the expression of a coding sequence inserted in a variety of cell types. The main disadvantages of using a retrovirus expression vector are (1) insertional mutagenesis, that is, the insertion of the therapeutic gene in an undesirable position in the target cell genome, which, for example, leads to unregulated cell growth and (2) the need for target cell proliferation for the therapeutic gene carried by the vector to be integrated into the target genome. Despite these apparent limitations, delivery of a therapeutically effective amount of a therapeutic agent via retrovirus can be effective if the efficiency of transduction is high and / or the number of target cells available for transduction is high.
[00107] Yet another viral candidate useful as an expression vector for the transformation of dermal mesenchymal stem cells is adenovirus, a double-stranded DNA virus. Like the retrovirus, the adenovirus genome is adaptable to use as an expression vector for gene transduction, that is, removing the genetic information that controls the production of the virus itself. Since adenovirus works normally in an extrachromosomal manner, recombinant adenovirus does not have the theoretical problem of insertional mutagenesis. On the other hand, adenoviral transformation of a target dermal mesenchymal stem cell may not result in stable transduction. However, more recently it has been reported that certain adenoviral sequences confer specificity of intrachromosomal integration to vehicle sequences, and thus result in a stable transduction of exogenous genetic material.
[00108] Thus, as will be evident to a person skilled in the art, a variety of suitable vectors are available to transfer the exogenous genetic material to dermal mesenchymal stem cells. The selection of an appropriate vector to deliver a therapeutic agent of a particular condition accessible to gene replacement therapy and optimization of the conditions for the insertion of the selected expression vector in the cell are within the scope of an ordinary specialist in the art without the need for experimentation excessive.
[00109] Thus, the present invention makes it possible to genetically engineer dermal mesenchymal stem cells in such a way as to produce polypeptides, hormones and proteins not normally produced in human stem cells in biologically significant quantities or produced in small quantities but in situations in which overproduction would lead to a therapeutic benefit. These products would then be secreted into the bloodstream or other areas of the body, such as the central nervous system. Human stem cells formed in this way and introduced into a support can serve as continuous drug delivery systems to replace the present regimens, which require periodic administration (by ingestion, injection, prolonged-release infusion, etc.) of the required substance . This invention has applicability in the supply of hormones, enzymes and drugs to humans, in need of such substances. It is particularly valuable in providing such substances, such as hormones (eg, parathyroid hormone, insulin), which are needed in sustained doses over extended periods of time and are associated with the tissue that is repaired.
[00110] ABCB5 (+) stem cells can be isolated to produce totipotent, multipotent or pluripotent stem cells (for example, induced pluripotent stem cells (iPSCs)), from which other cells, tissues and / or whole animals can develop. Thus, methods for direct reprogramming or induction of ABCB5 (+) stem cells to become totipotent, multipotent or pluripotent stem cells before or after the cells are seeded in the support, are provided in some aspects of the invention. The term "reprogramming", as used in this application, refers to a process that reverses the development potential of a cell or cell population (for example, an ABCB5 (+) stem cell). In this way, reprogramming refers to a process of directing a cell to a state with the potential for higher development, that is, back to a less differentiated state. The cell to be reprogrammed may be partially or definitely differentiated before reprogramming. In some modalities, reprogramming includes a complete or partial atavism of the state of differentiation, that is, an increase in the development potential of a cell, to that of a cell that has a totipotent, multipotent or pluripotent state. In some modalities, reprogramming involves the conduction of an ABCB5 (+) stem cell to a totipotent, multipotent or pluripotent state, such that the cell has the potential for the development of an embryonic stem cell, that is, a phenotype of embryonic stem cell. Reprogramming also encompasses the partial atavism of a cell's differentiating state to a state that makes the cell more susceptible to completing reprogramming to a totipotent, multipotent or pluripotent state when subjected to additional manipulations.
[00111] Totipotent, multipotent or pluripotent stem cells can be generated from ABCB5 (+) stem cells (referred to in this application as "reprogrammed ABCB5 (+) cells") using various reprogramming factors. The resulting cells, which have a greater development potential than ABCB5 (+) stem cells, can then become the source of stem cells for further manipulation. A "reprogramming factor", as used in this application, refers to a potential developmental change factor, the expression of which contributes to the reprogramming of a cell, for example, an ABCB5 (+) stem cell, to a less differentiated or undifferentiated state, for example, to a cell of a pluripotent state or partially pluripotent state. Reprogramming factors include OCT4, SOX2, KLF 4 and c-MYC (otherwise known as "Yamanaka factors"). Other reprogramming factors include, without limitation, SOX 1, SOX 3, SOX15, SOX 18, NANOG, KLF1, KLF 2, KLF 5, NR5A2, LIN28, 1-MYC, n-MYC, REM2, TBX3, TERT and LIN28. Any combination of two or more of the preceding transcription factors can be used to reprogram isolated ABCB5 (+) stem cells. Methods of reprogramming cells to a totipotent, multipotent or pluripotent state is described by Stadtfeld and Hochedlinger [33], incorporated in this application by reference in its entirety.
[00112] Differentiated cells can also be produced and incorporated into the support of reprogrammed ABCB5 (+) cells. The methods can understand the expression in the ABCB5 (+) cells reprogrammed one or more differentiation factors necessary to promote differentiation into a more mature, differentiated cell type such as, for example, a blood cell, platelet, stromal cell, cell bone, muscle cell, skin cell, neural cell or fat cell. As used in this application, the term "differentiating factor" refers to a potential developmental alteration factor such as a protein or small molecule that induces a cell to differentiate itself from a desired cell type, for example, a factor of differentiation reduces the potential for development of a cell. Differentiation to a specific cell type may involve the simultaneous and / or successive expression of more than one differentiation factor. The methods may further comprise the growth of reprogrammed ABCB5 (+) cells under conditions to promote differentiation to form a differentiated cell.
[00113] A "stem cell", as used in this application, is an undifferentiated or partially differentiated cell that has the capacity for self-renewal and has the potential for development to differentiate into multiple cell types. A "pluripotent cell" is a cell with the potential to develop, under different conditions, to differentiate itself from the characteristic cell types of the three layers of the germ cell, ie endoderm (eg, intestinal tissue), mesoderm (including blood, muscle and vessels), and ectoderm (such as skin and nerve). A "multipotent" cell is a cell that has the potential to develop into cells in one or more germ layers, but not all three. These cells include, for example, adult stem cells, such as, for example, hematopoietic stem cells and neural stem cells. A "totipotent" cell is a cell that has the potential for development to differentiate into all differentiated cells in an organism, including extraembryonic tissues. Stem cells can be prone to a differentiated phenotype; however, these cells can be induced to invert and re-express the stem cell phenotype. This process is referred to as "de-differentiation" or "reprogramming".
[00114] ACB5 (+) stem cells, reprogrammed ABCB5 (+) cells and differentiated cells of the invention can be manipulated under standard conditions for these cell types. The treatment of the cells can be carried out before or after the cells are incorporated into the support and in vitro, ex vivo or in vivo. For example, the cells can be present in the body or in culture medium. Manipulations can be performed under low or high oxygen conditions.
[00115] "Culture medium" contains nutrients that maintain cell viability and support proliferation. Typical culture medium includes: salts, buffers, amino acids, glucose or other sugar (s), antibiotics, serum or serum substitute and / or other components such as peptide growth factors. Cell culture media for use in deriving and maintaining totipotent, multipotent and pluripotent cells are known in the art. Culture medium may also include specific cell growth factors, such as angiogenin, bone morphogenic protein-1, bone morphogenic protein-2, bone morphogenic protein-3, bone morphogenic protein-4, bone morphogenic protein-5, bone morphogenic protein-6 , bone morphogenic protein-7, bone morphogenic protein-8, bone morphogenic protein-9, bone morphogenic protein-10, bone morphogenic protein-11, bone morphogenic protein-12, bone morphogenic protein-13, bone morphogenic protein-14, bone morphogenic protein-14, bone morphogenic-15, bone morphogenic protein IA receptor, bone morphogenic protein IB receptor, brain-derived neurotrophic factor, ciliary neurotrophic factor, ciliary neurotrophic factor-alpha, cytokine-induced neutrophil chemotactic factor 1, cytokine-induced neutrophil, neutrophil chemotactic factor 2-alpha, cytokine-induced chemotactic factor 2 beta, beta endothelial cell growth factor, endothelium 1, c factor epidermal termination, epithelium-derived neutrophil attractor, fibroblast growth factor 4, fibroblast growth factor 5, fibroblast growth factor 6 fibroblast growth factor 7, fibroblast growth factor 8, fibroblast growth factor b , fibroblast growth factor c, fibroblast growth factor 9, fibroblast growth factor 10, acidic fibroblast growth factor, basic fibroblast growth factor, strain-derived neutrophil factor alpha-1 receptor glial cell, alpha-2 receptor of the neutrophil factor derived from the glial cell line, growth-related protein, protein related to alpha growth, protein related to beta growth, protein related to gamma growth, epidermal growth factor binding to heparin, hepatocyte growth factor, hepatocyte growth factor receptor, failure-like growth factor line I, insulin-like growth factor receptor, insulin-like growth factor II, insulin-like growth factor binding protein, keratinocyte growth factor, leukemia inhibitory factor, leukemia inhibitory factor alpha receptor, nerve growth factor, nerve growth factor receptor, neurotrophin-3, neurotrophin-4, placenta growth factor, placenta growth factor 2, platelet-derived endothelial cell growth factor, growth factor platelet-derived growth factor, platelet-derived growth factor A, platelet-derived growth factor AA, platelet-derived growth factor AB, platelet-derived growth factor B, platelet-derived growth factor BB, alpha-platelet-derived growth factor receptor, beta-platelet-derived growth factor receptor, pre-B cell growth stimulating factor, stem cell, stem cell factor receptor, alpha growth transformation factor, beta growth transformation factor, transformation factor beta-1, growth transformation factor beta-1-2, transformation factor beta-2 , transforming factor beta-3, transforming factor beta-5, transforming factor beta-1 latent, growth transforming factor beta-l protein, transforming growth factor beta-ll protein, protein growth transformation factor beta-ill binding factor, tumor necrosis factor type I receptor, tumor necrosis factor type II receptor, urokinase type plasminogen activator receptor, vascular endothelial growth factor and chimeric proteins and fragments biologically or immunologically active.
[00116] The state of differentiation of the cell can be assessed using any method known in the art for making such assessments. For example, the differentiating state of a cell treated according to the methods described in this application can be compared to an untreated cell or cells treated with DNA using viral vectors to deliver DNA that results in the expression of the same differentiating or reprogramming factors.
[00117] The dose of stem cells can be defined by the number of cells included in the support and varies within wide limits and will naturally be adjusted to the individual requirements in each particular case. The number of cells used will depend on the weight and condition of the recipient and other variables known to those skilled in the art.
[00118] The present invention also provides some of the aforementioned compositions in kits, optionally including instructions for using the composition for treating a condition described in this application. That is, the kit may include a description for the use of the composition for participation in any biological or chemical mechanism described in this application. The kits may also include a description of the activity of the condition in the treatment of the condition, as opposed to the symptoms of the condition. That is, the kit can include a description for the use of the compositions as discussed in this application. The kit may also include instructions for using a combination of cells and support for treating diseases. Instructions can also be provided for administering the composition by any suitable technique. The kits can also be one or more reagents associated with the isolation and purification of dermal mesenchymal stem cells, that is, ABCB5 antibodies and instructions for isolating and / or purifying the cells.
[00119] The kits described in this application can also contain one or more containers, which can contain the composition and other ingredients as previously described. The kits may also contain instructions for mixing, diluting and / or administering the compositions of the invention in some cases. The kits can also include other containers with one or more solvents, surfactants, preservatives and / or diluents (for example, normal saline (NaCI 0.9%) or 5% dextrose) as well as containers for mixing, diluting or administering the components in a sample or an individual in need of such treatment.
[00120] The kit's compositions can be supplied as any suitable form, for example, as liquid solutions or as dry powders. When the composition provided is a dry powder, the composition can be reconstituted by adding a suitable solvent, which can also be supplied. In embodiments where the liquid forms of the composition are used, the liquid form can be concentrated or ready to use.
[00121] The present invention is further illustrated by the following Examples, which should in no way be construed as a limitation. The entire contents of all references (including literature references, issued patents, published patent applications and co-pending patent applications) cited in all parts of this patent application are hereby expressly incorporated by reference. EXAMPLES Example 1: Multipotent differentiation plasticity of ABCB5 + dermal stem cells in vitro and in vivo.
[00122] It has previously been shown that ABCB5 identifies a population of mesenchymal stem cells in the human dermis, where it confers hyperpolarization of the membrane and determines as a regulator of the membrane potential the propensity of skin parents to undergo differentiation. Additional studies revealed that ABCB5 confers resistance to drugs and marks subsets of cancer stem cells (CSC) with specific differentiation plasticity in human melanomas, where it also correlates with the progression of clinical disease.
[00123] ABCB5 + skin cells have been shown to reside in the reticular dermis and are distinct from neighboring mature fibroblasts, CD31 + endothelial cells and CD34 + bulge cells. ABCB5 is expressed by 2.5-5% of all cells in the human skin specimen. ABCB5 + cells coexpress the CD29 mesenchymal stem cell markers (in 99.48 ± 0.5% of cells), CD44 (99.09 ± 0.9%), CD49e (92.61 ± 4.0%), CD90 (100%), and CD166 (58.29 ± 19.7%), as well as the stem cell marker CD133 (6.29 ± 5.1%), but were negative for differentiation markers such as the CD31 marker of endothelial gem-line, hematopoietic CD45 marker and quiescent fibroblast CD34 marker. Importantly, only the distinct subpopulations of cells that positively target the reported MSC markers (CD29, CD44, CD49e, CD90 and CD 166) positively targeted ABCB5, while it was found that large proportions of cells expressing these antigens were negative for ABCB5, demonstrating that ABCB5 + cells represent a unique new subpopulation of mesenchymal stem cells.
[00124] The plasticity of multipotent differentiation of ABCB5 + dermal stem cells was compared with ABCB5 cells- in vitro and in vivo, in order to investigate whether ABCB5 represents a more specific marker of multipotent mesenchymal stem cells than antigens MSC currently available.
[00125] The differentiation potential of isolated ABCB5 + cells was examined by the positive selection of dissociated skin cell suspensions derived from healthy human volunteers and compared with the differentiation potential of ABCB5- dermal fibroblasts (Figure 1). ABCB5 + or ABCB5- cells were cultured in neurogenic, angiogenic, myogenic, osteogenic or adipogenic lineage induction media and their differentiation plasticity was assessed by measuring RNA induction and protein expression of specific markers for the lineage (ie , spectrin - myogenesis, CD31 - angiogenesis, TUJ1 - neurogenesis, Oil Red - adipogenesis, and Alizarin Red - osteogenesis), as well as morphological changes characteristic of the strain (Figure 1). Only ABCB5 + cells, but not ABCB5-dermal cells were able to give rise to the three embryonic strains (ie, ectodermal (neurogenesis), mesodermal (myogenesis) and endodermal (angiogenesis)) lines (Figure 1).
[00126] To further dissect the differentiation plasticity of human ABCB5 + dermal MSC and determine its ability independent of the multipotent differentiation niche in vivo, the myoregenerative potential of human ABCB5 + in vivo against skin-derived ABCB5 cells was examined in a lesion model of acute muscle. ABCB5 + and ABCB5- skin cells were injected into the anterior tibial (TA) muscles injured with severely immunocompromised NOD / SCID / IL2RY - / - (NSG) mice. Immunofluorescence staining representative of murine muscles injected with human ABCB5 + and ABCB5- cells with human-specific β2-microglobulin, Δ-sarcoglycan and spectrin was obtained. The cores are visualized with DAPI. Injected muscles and non-injected control muscles were collected 2 weeks after transplantation and examined for the expression of β2 human specific microglobulin (β2M), which identifies all cells of human origin and human specific spectrin (SPTBN1) and delta- sarcoglycan (SGCD), which are specifically expressed by differentiated but not murine human myocytes. Although immunostaining revealed the presence of human β2M + cells in both muscles injected with ABCB5 + and ABCB5- cells, indicative of successful transplantation and grafting, only TA muscles injected with ABCB5 + cells contained differentiated SPTBN1 + and SGCD + myocytes. Real-time PCR analyzes of injected and non-injected control muscles demonstrated the expression of human-specific β2M- transcripts in muscles injected with the ABCB5 + and ABCB5- cell, but not in uninjected controls, and the expression of SPTBN1 transcripts. and human specific SGCD was only demonstrated in muscles injected with the ABCB5 + cell. Thus, ABCB5 represents a highly specific marker of multipotent human dermal mesenchymal stem cells, with the function of a substantially increased discriminatory marker over the currently available MSC antigens. These findings highlight the ability of dermal MSC ABCB5 + as a new cell source for stem cell-based tissue regeneration. Example 2: Phenotype of Abcbõ knockout mouse stem cell deficiency.
[00127] In order to further dissect the role of ABCB5 in stem cell development and function, the first conditional Abcbõ (KO) knockout mouse was created. Until recently, the protein function of ABCB5 has been studied extensively in Homo sapiens. The human ABCB6 gene encodes an 812 amino acid (AA) protein with five transmembrane helices flanked by both extracellular and intracellular binding domains of ATP1. Previous studies revealed that one of the monoclonal anti-ABCB6 antibody clones, 3D2-1D12, which targets an extracellular loop containing amino acid residues 493-308 of the human ABCB5 protein, inhibits ABCB6-mediated efflux dye Rhodamine 123, polarization membrane and doxorubicin transport125, demonstrating the critical functional importance of this region of the molecule's extracellular loop. The corresponding ABCBõ mouse homolog, murine Abcbõ23, was also previously cloned. A homologous part of the murine molecule was targeted to disrupt Abcbõ's function in mice. Using the UCSC Blat search engine, it was identified that the mouse genomic region encoding the Abcbõ protein domain homologous to the 3C2-1D12 ABCBÕ mAb binding epitope is encoded by exon 23. Based on this finding, a conditional KO construct where two loxP sites were inserted to flank the murine exon 23 was designed (Figure 2).
[00128] To determine the result of a complete loss of function, exon 23 of the Abcbõ gene was deleted using the El-la-Cre transgene, which expresses Cre recombinase in a mouse embryo in the zygote stage 48 49. The deletion of the genomic region between loxP sites was confirmed by genomic DNA PCR. The heterozygous Abcbõnull / WT mice were intercrossed to produce homozygous Abcbõnull / null mutants, and the loss of Abcbõ expression and function in Abcbõnull / null mutants was confirmed by flow cytometric analyzes and Rhodamine-1231 efflux assays (Figure 3) .
[00129] Relative quiescence is one of the distinguishing characteristics attributed to various populations of mammalian stem cells 50. Skin stem cells were first identified as slow-cycle cells using so-called experimental "pulse and tracking DNA labeling approaches" "developed by Bickenbach 51, Morris 52 and Cotsarelis 50. These approaches depend on the incorporation of a labeled thymidine analog, uridine, in nuclear DNA during the replication phase (S phase) of the cell cycle. First, during DNA staining or the "pulse" phase, cells or animals are exposed to radioid or chemically labeled uridine (eg, bromodeoxyuridine, Brdll), which is then incorporated into nuclear DNA during each cell division. Exposure to labeled uridine (eg, Brdll) is then removed during the "screening" phase, with subsequent loss of the marker by frequent division of the cells, over the course of approximately 48 to 72 hours. Slow-cycle cells, however, are capable of conserving the DNA marker for extended periods of time; for example, stem cells from the lump area of the mouse hair follicle can conserve radiolabeled uridine for at least 4 weeks 50 and are therefore called "marker conservative cells"
[00130] In order to determine whether ABCBõ identifies a quiescent cell population that retains the marker on the mammalian skin, compatible with the demonstrated stem cell phenotype, and whether intact ABCB5 function is required for maintaining the stem cell quiescence , Brdll marking experiments were performed in Abcbõ WT and Abcbõ KO mice. Brdu is a non-radioactive urine derivative, which can be detected using fluorescently labeled anti-Brdll antibodies. The mice were subjected to a 9-day "pulse" of daily systemic BrdU administration (i.v.) designed to mark cells in slow division, followed by a Brdll-free "tracking" phase 4 weeks after the cessation of Brdll treatment. The percentage of Brdll retaining cells in dissociated full-thickness skin cell suspensions was then compared between Abcbõ WT and Abcbõ KO mice, using anti-Brdll antibody labeling and flow cytometry. The cells were thereby co-labeled with the dye 7-amino-actinomycin D (7-AAD), which binds to the total DNA, for enumeration and characterization of cells as to their cell cycle position (Figure 4). Flow cytometric analyzes revealed that after a 4-week "scan", all Brdu-positive (i.e., marker-retaining cells) in Abcbõ WT mice were found in the GO phase (Figure 4B, port R1), and that this population was decreased by 74% in Abcbõ KO mice (Figure 4C, port R1). In addition, suspensions of full-thickness skin cells derived from Abcbõ KO mice exhibited 67% more BrdU-negative cells proliferating in the S / G2 / M phase compared to those derived from Abcbõ WT mice (Figure 4B and 4C, port R2 ), indicating that the cancellation of normal ABCBõ function induces cell proliferation of normally quiescent ABCBÕ + cells.
[00131] Consistent with this observation, real-time PCR analyzes of the 84 cellular genes involved in cell cycle regulation (SABiosciences, catalog number PAMM-020), revealed significant negative regulation in 22-molecule Abcbõ KO mice involved in various canonical pathways of the cell cycle, including p53 signaling, G1 / S control regulation, cyclins and cell cycle regulation and calcium signaling pathways (Figure 5) including, members of the p53 family (p53 and p63) and the cKip family (p21 and p27), which control the control of the G0 / G1 cell cycle and cell quiescence.
[00132] These results indicate that ABCB5 regulates the progression of the cell cycle and is required for maintaining the quiescence of the stem cell. The abolition of ABCB5 function leads to the repression of negative regulators critical to the progression of the G0 / G1 cell cycle and to increased cell proliferation. The withdrawal of the cell cycle is a prerequisite for normal differentiation, and the inability to do so may explain impairment of normal wound healing in Abcbõ KO mice described below. The data further support the convenience of ABCB5 as a functionally relevant marker for the isolation of the stem cell from mammalian skin. Example 3: Impaired wound healing in Abcbõ knockout mice.
[00133] Wound healing is a complex phenomenon that progresses through four sequential phases: hemostasis, inflammation, proliferation, and remodeling with scar formation 53. It was investigated whether the function of intact ABCB5 is required for normal wound healing , using Abcbõ WT and Abcbõ KO mice. Full-thickness skin wounds were generated by removing 1 cm2 of skin and panicle, and the mice were subsequently observed for 7 days during the first proliferative phase of wound healing. The wounds were photographed immediately after the surgical procedure and at the time of tissue collection (day 7). The digital photographs captured at the end of the experiment were quantitatively analyzed in comparison with the corresponding initial photographs by 2 independent observers blinded to the genetic status of the mouse.
[00134] Wound closure was hereby calculated as a percentage of the original wound, based on measurements obtained by planimetric analysis as previously described 54, using the Image J software package (NIH, Bethesda, MD). The data were compared using unpaired t-tests with Welch correction to account for uneven variations. Abcbõ KO mice (n = 15) showed significantly delayed wound closure compared to Abcbõ WT mice (n = 19) (63.64 ± 7.6% versus 87.33 ± 3.4%, mean ± SEM, P = 0.01) (Figure 6). Additionally, the thickness of the inflammatory stroma was measured in both groups in the scanned wound cross sections, marked with H&E, from day 7 using the Aperio Image Scope program (Vista, CA), with Abcbõ KO's wounds showing significantly increased thickness of the inflammatory stroma compared to the wounds of Abcbõ WT (820.2 ± 65.6pm versus 590.0 ± 38.8 pm, mean ± SEM, P = 0.003) (Figure 6).
[00135] Neovascularization is one of the marks of the early stages of wound regeneration and is critical for the control of hypoxia-induced excessive myofibroblast proliferation, which is believed to contribute to the formation of the keloid scar (reviewed in 55). Previous studies have shown that although wound angiogenesis is primarily achieved by growing new vessels from pre-existing vessels, some newly formed vessels could originate from bone marrow progenitor cells56. The contribution of other resident stem cell populations to wound angiogenesis is unclear. Based on our previous observation of the critical role of ABCB5 + cells in the vasculogenesis of human melanoma28, it is assumed that the function of intact ABCB5 could also be essential for efficient angiogenesis in injured skin. To test this hypothesis in additional preliminary studies generated since the last iteration of this proposal, the formation of vessels in murine skin wounds generated as described above was analyzed by comparing the expression of the endothelial CD31 marker in Abcbõ KO and Abcbõ WT mice on day 7 after the wound in the context of the thickness of the vascularized versus avascularized strata (layers) within the wound bed. Vascular CD31-positive and avascular CD31-negative layer thicknesses were measured in both groups in scanned wound cross sections, marked with CD31, on day 7 using the Aperio Image Scope program (Vista, CA), with Abcbõ wounds KO showing significantly reduced thickness of the vascular layer (278.5 ± 51.45 pm versus 469.2 ± 40.30 pm, mean ± SEM, P = 0.0069) and significantly increased thickness of the avascular layer (626.2 ± 41 , 18 pm versus 324.5 ± 28.55 pm, mean ± SEM, P <0.0001) compared to the Abcbõ WT wound (Figure 7).
[00136] In contrast to wild animals, where the iterative CD31-positive vessels variably formed elongated branched channels with well-formed lumens, similar areas of KO ABCB5 animals were relatively devoid of CD31-positive structures or were composed of profiles occasional small vessels mixed with solitary or small groups of CD31-positive cells. These last CD31-positive cells seemed to reflect the inability to differentiate into tubules capable of angiogenic budding necessary to form the branching network of mature vessels required for an efficient and productive healing response (Figure 8).
[00137] Consistent with this observation, real-time PCR analyzes of the 84 genes involved in angiogenesis (SABiosciences, catalog number PAMM-024Z), revealed significant negative regulation in pro-angiogenic cytokine wounds, that is é, Csf3, CxCI2, 111 b, Ifng, Lep, and Mdk and positive regulation of a known antiangiogenic molecule, phosphatidylserine Bail receptor (Figure 9), providing an initial explanation for the highly inhibited and abnormal angiogenic model observed in Abcbõ KO wounds .
[00138] These results show impaired wound healing in Abcbõ KO mice characterized by delayed wound closure, increased inflammatory stroma thickness and aberrant angiogenesis, still supporting a critical functional role of ABCB5, and therefore of ABCB5 + mesenchymal stem cells, in normal healing of skin wounds. Example 4: Effect of human dermal ABCB5 + MSC on regenerative wound healing in NSG mice.
[00139] The effect of human ABCB5 + dermal MSC on wound healing in NSG mice treated with INTEGRA® was investigated using the following four treatment groups: (1) no treatment; (2) INTEGRA® only; (3) INTEGRA® injected with 1x106 ABCB5 + dermal MSC cells and (4) INTEGRA® injected with 1x106 ABCB-positive dermal cells (Figure 10B). Full-thickness skin wounds were generated in all animals by removing 1 cm2 of skin and panicle as described in Figure 6. Each wound in groups 2, 3 and 4 was immediately transplanted with a 1 cm2 graft of INTEGRA®, followed by intra injection -INTEGRA® of 1x106 ABCB5 + cells (group 3) or 1x106 ABCB5- cells (group 4). No cells were injected in experimental group 2. Group 1 served as an experimental control without treatment. On day 14 after the procedure, wound tissues, consisting of INTEGRA®, surrounding skin and underlying muscle tissue, were collected. The thickness of the inflammatory stroma was measured in all groups in the scanned wound cross sections, marked with H&E, on day 14 using the Aperio Image Scope program (Vista, CA). Quantitative analyzes revealed that mice treated with INTEGRA® and human ABCB5 + dermal MSC exhibited significantly reduced inflammatory stromal thickness compared to mice treated with INTEGRA® and ABCB5-dermal cells (608.0 ± 46.7 pm versus 855.4 ± 69.8 pm, mean ± SEM, P = 0.009), mice treated with INTEGRA® alone (874.7 ± 43.3 pm, P = 0.0001), or untreated mice (1014 ± 49.4 pm, P <0.0001). No difference was observed between mice treated with INTEGRA® alone and mice treated with INTEGRA® and ABCB5- dermal cells. INTEGRA® treatment only modestly reduced the thickness of the inflammatory stroma compared to untreated wounds (Figure 10A). To ensure the viability of the injected human cell population, the grafts, moreover, were examined for the expression of human-specific β2 microglobulin (β2M), an identifier of all cells of human origin. Immunomarking revealed the presence of groups of human β2M + cells as well as individual human β2M + cells (Figure 10C) in INTEGRA® grafts injected with cells. Real-time PCR analyzes of INTEGRA® grafts injected with ABCB5 + or human ABCB5-dermal cells also demonstrated the expression of human specific GAPDH and β2M transcripts, which were not detectable, as expected, in controls treated with INTEGRA® only. not injected (Figure 10D). Thus, despite the successful graft and persistence of viable ABCB5 + dermal MSC or viable ABCB5-dermal cells after injection into transplantable INTEGRA® matrices, only ABCB5 + dermal MSC transplantation, but not human ABCB5-dermal cells, further reduced the thickness inflammatory stroma in injured NSG mice, providing proof of the initial principle of the specific therapeutic utility of this new MSC population to further increase regenerative wound healing. Example 5: Humanized mouse model
[00140] Although mouse models are widely used for the study of human disease, there are substantial differences in the wound healing process between human and non-human species31. For this reason, an alternative model in which full-thickness human skin grafts are transplanted into immunodeficient mice32 was used. In this model, skin grafts closely resemble human skin histologically and maintain their human phenotype for at least 3 months. Samples of normal adult skin discarded removed during plastic surgery were used for human skin grafting to immunodeficient NSG mice at 8 weeks of age (Figure 11 A, B). The human skin xenograft wound resulted in a normal human wound healing model as shown in Figure 11C.
[00141] In addition, the effects of INTEGRA® support transplantation on regenerative wound healing in human patients were examined on biopsies obtained from non-grafted wounds (Figure 12, upper panels), or from human patient wounds transplanted with support grafts. INTEGRA® (Figure 12, bottom panels). Comparative analyzes revealed immunohistochemical profiles of healing responses resulting in scar formation (no INTEGRA®, Figure 12, upper panels) or support-induced regeneration, characterized by the realignment of myofibroblasts that express actin and markedly increased the expression of dermal cells ABCB5 + in wounds that carry the support (INTEGRA®, Figure 12, lower panels).
[00142] These results show that the INTEGRA® support graft and the resulting mobilization of ABCB5 + dermal MSC preferentially induce regenerative wound healing as opposed to wound healing via scar formation. Thus, human ABCB5 + dermal MSC significantly and selectively increase INTEGRA®-mediated regenerative wound healing (Figure 10) and demonstrate the therapeutic efficacy of the human ABCB5 + co-graft from the dermal MSC / INTEGRA® support in wound healing experiments most relevant human skin translationally.
[00143] Dermal mesenchymal stem cells must provide a balance whereby normal healing responses occur in an orderly manner, resulting in gradual wound closure and physiological scar formation. It is expected that impaired dermal stem cell function in Abcbõ KO mice will lead to abnormal wound healing responses, which may be insufficient or excessive scarring. Poor healing can be manifested as loss of subcutaneous tissue as seen in decubitus ulcer or failure of re-epithelialization as seen in venous ulcer or combination of necrosis infection as seen in diabetic ulcer. Histologically, deficient wound healing can be characterized by ex-cessive and increased neutrophil infiltration of MMP-9 collagenase secretion that leads to collagen destruction 3. In contrast, hypertrophic scars are characterized by increased collagen deposition due to a response inflammatory disease amplified with overproduction resulting from growth factors, including TGF-β58. 1. Frank, N.Y. et al. 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[00144] All references cited in this application are fully incorporated by reference. Having thus described various aspects of at least one embodiment of this invention, it should be appreciated that various changes, modifications, and improvements will occur promptly to those skilled in the art. Such changes, modifications and improvements are intended to be part of this disclosure and are intended to be within the spirit and scope of the invention. Consequently, the preceding description and drawings are by way of example only.

权利要求:
Claims (12)
[0001]
1. Support for wound healing, characterized by the fact that it comprises a support of collagen glycosaminoglycan co-grafted with a population of stem cells, in which the population of stem cells is: a) at least 80% of the population of stem cells stem cells are ABCB5 + stem cells, b) the stem cell population is ocular ABCB5 + stem cells, where the cell population is free of non-ocular cells, or c) ABCB5 + stem cells are isolated from an individual's tissue , in which ABCB5 + stem cells were separated from other cells in the individual using an antibody specific to ABCB5.
[0002]
2. Support according to claim 1, characterized by the fact that at least 85% or 90% of the stem cell population are ABCB5 + stem cells.
[0003]
3. Support according to claim 1 or 2, characterized by the fact that the support is a porous matrix of cross-linked collagen and glycosaminoglycan and in which the collagen, optionally, is the bovine tendon collagen.
[0004]
4. Support according to any one of claims 1 to 3, characterized in that the support includes a semipermeable layer, which is optionally polysiloxane (silicone).
[0005]
Support according to any one of claims 1 to 4, characterized in that the support is a network support.
[0006]
6. Support according to any one of claims 1 to 5, characterized in that the support has a pore size of 10 to 500 micrometers, 50 to 350 micrometers, or 70 to 200 micrometers.
[0007]
Support according to any one of claims 1 to 6, characterized by the fact that it further comprises at least one bioactive molecule effective in increasing wound healing.
[0008]
Support according to any one of claims 1 to 7, characterized in that it is for use in promoting wound healing, wherein a wound is contacted with the support for wound healing to promote healing.
[0009]
9. Support according to claim 8, characterized by the fact that the wound is a burn or a diabetic ulcer.
[0010]
10. Support according to claim 8, characterized by the fact that negative pressure healing therapy is used with the support.
[0011]
11. Support for wound healing, characterized by the fact that it is for use in promoting wound healing, in which a wound is brought into contact with the support for wound healing, in which the support comprises a support of collagen glycosaminoglycan co-grafted with a population of ABCB5 + stem cells, and in which ABCB + stem cells are isolated from the eye, where the cell population is free of non-ocular cells.
[0012]
12. Wound healing support for use according to claim 11, characterized by the fact that the wound is an eye wound.

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同族专利:

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公开号 | 公开日

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KR20160042816A|2016-04-20|

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KR102319899B1|2021-11-01|

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EP2994173A1|2016-03-16|

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AU2014262590B2|2018-05-10|

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US20160106782A1|2016-04-21|

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CN105517587B|2019-02-19|

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HK1222586A1|2017-07-07|

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BR112015028193A2|2017-07-25|

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EP2994173B1|2021-07-07|

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AU2014262590A1|2015-12-03|

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WO2014182994A9|2015-03-26|

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MX2015015536A|2016-08-08|

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DK2994173T3|2021-10-11|

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CA2911232C|2021-11-09|

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EP2994173A4|2016-12-21|

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WO2014182994A1|2014-11-13|

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ES2892403T3|2022-02-04|

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CN105517587A|2016-04-20|

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JP6600299B2|2019-10-30|

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JP2016520068A|2016-07-11|

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CA2911232A1|2014-11-13|

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法律状态:
2018-02-27| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2019-07-30| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

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2020-04-22| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2020-10-06| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 09/05/2014, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

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申请号 | 申请日 | 专利标题

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US201361822134P| true| 2013-05-10|2013-05-10|

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US61/822,134|2013-05-10|

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PCT/US2014/037435|WO2014182994A1|2013-05-10|2014-05-09|Wound healing and tissue engineering|

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