OA19580A - Methods for development and use of minimally polarized function cell microaggregate units in tissue applications using LGR4, LGR5 and LGR6 expressing epithelial stem cells - Google Patents
Methods for development and use of minimally polarized function cell microaggregate units in tissue applications using LGR4, LGR5 and LGR6 expressing epithelial stem cells Download PDFInfo
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- OA19580A OA19580A OA1201800075 OA19580A OA 19580 A OA19580 A OA 19580A OA 1201800075 OA1201800075 OA 1201800075 OA 19580 A OA19580 A OA 19580A
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Abstract
Provided herein are constructs of micro-aggregate multicellular, minimally polarized grafts containing Leucine-rich repeat-containing G-protein coupled Receptor (LGR) expressing cells for wound therapy applications, tissue engineering, cell therapy applications, regenerative medicine applications, medical/therapeutic applications, tissue healing applications, immune therapy applications, and tissue transplant therapy applications which preferably are associated with a delivery vector/substrate/support/scaffold for direct application.
Description
MethodsJ^^^
Expressinq. Epithelial Stem Cells
Priority Claim
This application is a divisional of OAPI Patent Application No. 1201700207 filed 1 June 2017, which is based on PCT international application PCT/US2015/063114 filed 1 December2015, which claims the priority of U.S. Serial No. 14 /954335 filed 30 November 2015 and U.S. Provisional Application Serial No. 62/086,526 filed 2 December 2014, ail of which are hereby incorporated by reference.
Technical Field
The présent invention relates to constructs of micro-aggregate multicellular grafts containing Leucine-rich repeat-containing G-protein coupled Receptor (LGR) expressing cells for wound therapy applications, tissue engineering, cell therapy applications, regenerative medicine applications, medical/therapeutic applications, tissue healing applications, immune therapy applications, and tissue transplant therapy applications.
More particularly, the invention provides a deliverable micro-aggregate multicellular LGR construct on a delivery vector/substrate/support/scaffold for direct application.
Background
Over the years, clinicians and researchers hâve searched for antimicrobial agents that not only reduce microorganism wound burden but also possess less cytotoxic side effects. From burns to both acute and chronic wounds, there is the potential for manipulation of naturally-occurring, 25 self-derived antimicrobial peptides, in that these agents typically function through membrane permeabilization, a mechanism less îikely to lead to microbial résistance. With the continued risk of infections in wounds and the advancing épidémie of bacterial résistance to current antibiotic thérapies, there is a genuine need for the development of a new class of topical 30 antimicrobial agents for use in cutaneous burns and wounds.
There are essentially four phases of wound healing that hâve been described over the past century: (1 ) hemostasis, (2) inflammatory, (3) proliférative, and (4) remodeling. These sequential phases were first defined by the types of cells that had mïgrated into the wound and then later by the
..........................type of cytokines and growth factors expressed within the tissues.
With the recent progress in mesenchymal and adipose-derived stem cell isolation and transplantation, researchers hâve begun to study how these cells improve healing and alter expression within each stage, particularly throughout the later inflammatory to remodeling phases. Much like the mesenchymal and adipose-derived stem cells of the deeper compartiment, the épithélial stem cell develops from the primordial ectoderm, which later develops the more superficial épithélial compartment and, thus, also has a potential rôle in cutaneous wound healing. At this time, there is io limited research on how transplantation and application of isolated LGR4, LGR5 and LGR6 expressing épithélial stem cells alter wound healing gens expression.
It is known that LGR4, LGR5 and LGR6 expressing épithélial stem cell populations are often destroyed following severe full-thickness damage to is the skin, leaving tissues incapable of producing a viable and self-sustaining épithélial compartment. Despite a combination of granulatory and fibrotic efforts driven by localized inflammation and subséquent chemotaxis of a spectrum of cellular entitîes, without the épithélial stem cell focal niche, remaining tissues are left without the regenerative potential to form a functional epithelium, hair folîicle, sweat gland, or the like.
Complex fuit thickness injuries to human and mammalian tissues and/or complex injuries involving multiple tissue éléments (skin, muscle, fat, blood vessels, nerves and bone) are difficult in nature to heal. Such injuries and subséquent resulting wounds are also difficult to treat through current wound care methods, surgical interventions with current approved technologies utilizing cells, tissues, devices, biologics, drugs and/or growth factors. A common reason for such difficulty is that the tissues remaining in or around a wounded or injured tissue bed are typically devoid of inter-dependent, necessary components: 1 ) cellular progenitor and/or stem cell populations; 2) so extracellular matrix/scaffolding éléments and substrates; and 3) a combination of interactions between and among cellular entitîes and substrates. Such deficiency in the cellular niche, ECM (extracellular matrix) scaffolding and related interactive interfaces subsequently results in failure to re-generate or generate the essentiel multi-dimensional architecture required for cellular migration, différentiation, and tissue polarization. Without these cellto-cell and cell-to-matrix interactions, remaining cellular entities within the wound bed, no matter their proliférative or lineage potential, are forced to provide primarily a barrier utility ratherthan develop a more complex, multitissue construct capable of recognizable “function.” Consequently, the wound -whether involving skin, muscle, fat, tendon, bone--becomes subsequently scarred, disorganized and dysfunctional.
Current applications in field of tissue engineering of cultured skin, 10 cartilage, bone, muscle, blood vessels, nerves, lymphatics and related substitutes are largely based on a three part strategy: 1) acquiring a tissue source and harvesting cell suspension from such tissue; 2) applying these cells to a matrix or scaffold; and 3) grafting the construct onto or into a target site of a human or animal. However, in the absence of the above-identified inter15 dépendent, necessary components, tissue engineering applications, cell therapy applications, regenerative medicine applications, tissue healing applications and tissue transplant therapy applications do not possess the natural cellular micro-aggregate architecture needed to competently assemble functional polarized tissues. Thus, due to the lack of proper inter-dependency, 20 progenitor cell mass and proper scaffolding prevent such constructs to be useful in therapeutic applications such as multi-compartment tissue régénération and/or bone and muscle reconstruction.
Consequently, in part due to the foregoing, substantial efforts and resources hâve been directed by both industry and academies to developing 25 synthetic tissue substitutes, autograft constructs, as well as patient-derived epidermal expansion autografts (i.e. EPICEL® from Vericel Corporation of Cambridge MA.). These products, although bénéficiai, are often expensive and do not provide the patient with a true multi-compartment tissue construct. For example, cultured épithélial autograft (CEA) remains unable to restore both 30 épithélial and dermal compartments seen in native skin. But in view of the absence of interdependent functioning compartments, the cultured cells are left without an expanding localized stem cell population and the evolving tissue polarization needed to develop integument -- epidermis, dermis, glands and hair -- which truly defines skin. This failure, in turn, leads to monolayer fragility, épithélial instability, barrier breakdown, and scar.
Alternatively, the more robust acellular matrices such as ALLODERM® from LifeCell Corporation, INTEGRA® from Integra LifeSciences Corporation and DERMAMATRIX® a product from Musculoskeletal Transplant Foundation, although excellent reconstructive options, lack those properly placed lineage spécifie stem cell populations which are necessary to develop functional native tissues.
The inventor herein has already written about the relatively recent récognition of LGR5 and LGR6 as markers of both intestinal and epidermal stem cells in mammals. In Stimulation ofthe Follicular Bulge LGR5+ and LGR6+ Stem Cells with the Gut-Derived Human Alpha Defensin 5 Results in Decreased Bacterial Presence, Enhanced Wound Healing, and Hair Growth from Tissues Devoid of Adnexal Structures, Plast. Reconstr. Surg. 132: 1159, 2013, Leucine15 rich repeat-containing G-protein-coupled receptor (LGR) is a seven-pass transmembrane protein receptor with signifïcant sequence and structural homology to the follicle-stimulating hormone, thyroid-stimulating hormone, and luteinizing hormone receptor families.
In that study, it was recognized that human alpha defensin 5 peptide significantly enhanced wound healing and reduced basal bacterial load compared with human beta defensin 1 and sulfadiazine. Human alpha defensin 5 was the only therapy to induce LGR stem cell migration into the wound bed. In addition, gene heat mapping showed signifïcant mRNA up-regulation of key wound healing and Wnt pathway transcripts such as Wnt1 and Wispl. So it was concluded that human alpha defensin 5 could be used for enhanced wound healing due to the observed increase of LGR stem cell migration into wound beds and associated bacterial réduction and hair production through the augmentation of key Wnt and wound healing transcripts. In short, this and other work led to the récognition ofthe potential for using LGR4+, LGR5+ and LGR6+ expressing épithélial stem cells in direct biomédical engineering soft tissue constructs.
Summary of Invention
The invention provides in a first embodiment a minimally polarized micro19580
-...... aggregate multi-cellular composition including isolated Iiving LGR expressing........... cells and a multi-dimensionai support selected from the group consisting of scaffolding, collagen, matrix, particle, and fiber.
The invention provides in a further embodiment to the previous 5 embodiment a minimally polarized micro-aggregate multi-cellular composition including isolated living LGR expressïng cells and a multi-dimensionai support selected from the group consisting of scaffolding, collagen, matrix, particle, and fiber where the LGR expressing cells are supplemented with growth factors and where the LGR expressing cells are selected from the to group consisting of LGR4, LGR5 and LGR6.
The invention provides in a further embodiment to any of the previous embodiments a minimally polarized micro-aggregate multi-cellular composition including isolated living LGR expressing cells and a multidimensionai support selected from the group consisting of scaffolding, is collagen, matrix, particle, and fiber where the LGR expressing cells are supplemented with migratory/recruiting analytes and the LGR expressing cells being selected from the group consisting of LGR4, LGR5 and LGR6.
The invention provides in a further embodiment to any of the previous embodiments a minimally polarized micro-aggregate multi-cellular composition 20 including isolated living LGR expressing celte and a multi-dimensionai support selected from the group consisting of scaffolding, collagen, matrix, particle, and fiber where the LGR expressing cells are supplemented with LGR spécifie binding éléments selected from the group consisting of ligand familïes, Rspondin, EDGF, PDGF, Wnt, VEGF, and antimicrobial peptides and where the
LGR expressing cells are selected from the group consisting of LGR4, LGR5 and LGR6.
The invention provides in a further embodiment to any of the previous embodiments a minimally polarized micro-aggregate multi-cellular composition including isolated living LGR expressïng cells and a multi-dimensionai support 30 selected from the group consisting of scaffolding, collagen, matrix, particle, and fiber where the composition is used as a therapeutic construct for a select target consisting of a tissue région, wound, void, defect tissue, or blood for alteration of either surrounding adjacent tissues.
The invention provides in a further embodiment to any of the previous embodiments a minimally polarized micro-aggregate multi-cellular composition characterized by a isolated lïving LGR expressing cells transplanted to damaged tissue to accelerate healing thereof.
The invention provides in a further embodiment to any of the previous embodiments a minimally polarized micro-aggregate multi-cellular composition for tissue system repair or restoration throughout the body comprising a support scaffolding with isolated LGR containing cells secured thereto.
The invention provides in a further embodiment to any of the previous to embodiments a tissue graft for application to ectodermal, mesodermal or endodermal-derived tissues Systems throughout a mammalian body
Another embodiment of the invention is characterized by a method for obtaïning a minimally polarized micro-aggregate multi-cellular composition characterized by the steps of growing and isolating living LGR expressîng cells 15 for transplantation to a select mammalian target tissue.
The invention provides in a further embodiment to the foregoing method a method for obtaining a minimally polarized micro-aggregate multi-cellular composition characterized by the steps of growing and isolating living LGR expressing cells for transplantation to a select mammalian target tissue further characterized by the step of affixing the isolated living LGR expressing cells to a multï-dimensional support selected from the group consisting of scaffolding, collagen, matrix, particle, and fiber.
The invention is characterized in still another embodiment by a method for obtaining a minimally polarized micro-aggregate multi-cellular composition 25 characterized by the steps of growing and isolating living LGR expressing cells for transplantation to a select mammalian target tissue further characterized by the step of selecting the LGR expressing cells from the group consisting of LGR4, LGR5 and LGR6.
The invention provides in a further embodiment to any of the previous method 30 embodiments the step of applying the minimally polarized micro-aggregate multicellular to one of the group consisting of épithélial Systems, glands, hair, nerves, bone, muscle, fat, tendons, blood vessels, fascia, ocular tissues and peptide secreting cellular éléments employing delivery by a technique selected from the
group consisting of application, transplantation, implantation, directed seeding, directed migration, directed tracking, in setting, laminating and/or injection ofthe cellular element generating, regenerating, enhancing and healing.
The invention provides in a further embodiment to any of the previous method s embodiments a method for obtaining a minimally polarized micro-aggregate multi-cellular composition characterized by the steps of growing and isolating living LGR expressing cells for transplantation to a select mammalian target tissue further characterized by the step of applying the minimally polarized microaggregate multi-cellular composition directly to a tissue in vivo for tissue to restoration.
The invention provides in a further embodiment to any of the previous method embodiments a method for obtaining a minimally polarized micro-aggregate multi-cellular composition characterized by the steps of growing and isolating living LGR expressing cells for transplantation to a select mammalian target
I5 tissue further characterized by the step indirectly applying the minimally polarized micro-aggregate multi-cellular composition via the blood stream for tissue restoration in a body.
The invention ïs characterized in yet another embodiment by a method for producing a minimally polarized micro-aggregate multi-cellular composition 20 characterized by the steps of:
a) obtaining a tissue specimen;
b) extracting minimally polarized functional units containing LGR expressing cells from the specimen;
c) processing of hypodermis and subdermal fat cellular components from an 25 appropriate source;
d) adding the processed hypodermis and subdermal fat components to the extracted minimally polarized functional units to create épithélial stem cell singularity units;
e) enriching the épithélial stem cell singularity units;
f) adding the épithélial stem cell singularity units to a construct scaffold; and g) verifying the maintenance of minimum polarization of the obtained composition.
The invention provides in a further embodiment to any of the previous embodiments a media formulation used m obtaining minimally polarized micro-aggregate multi-cellular compositions using cell sustaining media composition for reducing the viability of micro-organisms during transport and processing of tissues, characterized by: a) a mixture of épithélial cells and kératinocytes; b) at least one agent selected from the group consisting of penicillin, streptomycin, and amphotericin B; and c) fibrinogen.
The invention provides in a further embodiment to the previous embodiment a cell sustaining media composition for reducing the viability of micro-organisms during transport and processing of tissues, characterized by: a) a mixture of épithélial cells and kératinocytes; b) at least one agent selected from the group consisting of penicillin, streptomycin, and amphotericin B; and c) fibrinogen, where the fibrinogen is human and where the agent includes both an antibiotic and an antimycotic for stabilizing human tissues.
In the context of a first aspect of the invention, it is characterized by LGR expressing cells being applied to scaffolding matrix, and/or fiber to thereby establish micro-aggregate multi-cellular grafts for tissue engineering applications, cell therapy applications, regenerative medicine applications, medical/therapeutic applications the grafts being directly applied to tissue or blood for improvement and or alteration of épithélial Systems throughout the body.
A second aspect of the invention is characterized by LGR expressing cells being applied to scaffolding, matrix, and/or fiber with/or without additional enhancing factors or analytes before or after either being applied to tissue or blood for improvement and or alteration of in vivo épithélial Systems.
A further aspect of the invention is characterized by LGR expressing cells altered by enhancing factors or analytes, being applied as targets within the body, tissue or blood for improvement and/or alteration of épithélial Systems through local or distant migration throughout the body and/or to restore gland and hair growth.
A fourth aspect ofthe invention is characterized by transplanting LGR expressing cells from tissue, blood or culture for alteration of surrounding adjacent tissues or distant tissues such as but not inclusive of LGR expressing cells applied to scaffolding, matrix, and fiber before or after either being applied to tissue or blood for improvement and or alteration of ectodermal, mesodermal or endodermal- derived tissues Systems throughout the body.
A fifth aspect of the invention is characterized by directly applying LGR 5 expressing cells to a delivery substrate vehicle selected from a group consisting of scaffolding, matrix, and fiber with/or without additional enhancing factors or analytes before or after either being applied to tissue or blood for improvement and or alteration of ectodermal, mesodermal or endodermalderived tissues Systems throughout the body.
Still another aspect of the invention is characterized by combining LGR expressing cells altered by enhancing factors or analytes, with a delivery support substrate as targets within the body, tissue or blood for improvement and or alteration of ectodermal, mesodermal or endodermal-derived tissues Systems throughout the body through local or distant migration throughout the 15 body.
A further aspect of the invention is characterized by adhering LGR expressing cells to a support substrate for the delivery, application, transplantation, implantation, directed seeding, directed migration, directed tracking, in setting, laminating and/or injection of the cellular element generating, regenerating, enhancing and/or healing épithélial Systems, glands, hair, nerves, bone, muscle, fat, tendons, blood vessels, fascia, ocular tissues and peptide secreting cellular éléments.
A final stated aspect of the invention is to generate LGR expressing stem cells as micro-aggregate multi-cellularfunctional units exhibiting minimal 25 polarization for transplantation and direct application to a target within a mammalian body, tissue or blood to enhance and accelerate tissue generating, regenerating, enhancing and/or healing.
In most general terms, the invention herein contemplâtes the transplanting and/or delivery of isolated LGR expressing cells (Leucine-rich 30 repeat-containing G-protein coupled receptor) for the génération, régénération, recruitment or enhancement of an épithélial system, hair, gland bone. The invention also contemplâtes application to both local/proximate and distant/remote tissue in clinical medicine, bioengineering and/or research
ΙΟ constructs using a delivery vehicle in the form of scaffolding, matrix orfiber with or without the supplémentation of growth factors migratory/recruiting analytes or LGR spécifie binding éléments such as but not limited to ligand families: R-spondin, EDGF, PDGF, Wnt, VEGF, antimicrobial peptides.
Use ofthe LGR épithélial stem cells, particularly in conjunction with a formed scaffolding substrate, provides full thickness wounds and or voids in épithélial Systems with a stem cell enriched tissue substitute. Moreover, the addition of this minimally polarized functional cell unit (MPFU) to an épithélial system enhances/improves the status of that epithelium which includes the growth, génération or régénération of hair, glands, secreted anti-microbial peptides, growth factors and analytes generally required to maintain and promote the health and viability of the epithelium and local surrounding tissues éléments.
Recognizing that LGR4+, LGR5+ and LGR6+ stem cell and progenitor cell prolifération kinetics remain high, especially when in contact with substrate scaffolding, complété épithélial turnover rates are typically less than 12 days (1cm inter-population distance spacing). This capacity to regenerate a sufficient tissue bi-layer and subséquent barrier function suggests a rôle for these cells as a type of evolving biologie dressing for complex full thickness and multi- tissue wounds.
Beyond a capacity to regenerate skin, muscle and bone quickly, the progenitors of the LGR4, LGR5 and LGR6 stem cells also hâve the ability to generate native anti-microbial peptides that not only reduce the basal level of microorganisms within the wound bed but also augment progenitor cells amplification and différentiation, leading to a réduction in wound and periwound infections, faster wound closure, and hair follicle development.
The invention herein describes the translational applicability of an LGR expressing épithélial stem cell -seeded scaffold in providing an immédiate, deliverable and viable tissue barrier that is capable of maintaining a stem cell colony focus with concomitant competent progeny. From these stem cell foci, progeny can undergo migratory proliferative-differentiation in order to stimulate épithélial tissue éléments, healing and graft intégration. It has been found that the LGR épithélial stem cells can be applied alone, with
.....-- scaffolding, soluble growth factors and/or additional cell lineage which promote the polarization ofthe scaffold bound populations as well as intrinsic tissue architecture required in épithélial healing and cellular regenerative efforts.
Broadly defined, the protocol of the invention involves: a) harvesting living human/mammalian tissue; b) processing th© tissue element to generale a micro-aggregate multi-cellular functional units which contain LGR expressing cells; c) applying the LGR expressing cell micro-aggregate multi-cellular functional units to a delivery vehicle substrate selected from the group ίο consisting of scaffolding, matrix, particle, cell(s) and fiber to croate a construct; d) optionally including selected additional enhancing factors; and e) applying the construct to tissues for generating, regenerating, enhancing and/or healing tissue Systems including those related to ectodermal, mesodermal and/or endodermal origin tissues including but not limited to skin. glands, hair, nerves, bone, muscle, fat, tendons, blood vessels, fascia, ocular tissues, bone marrow, lung, heart, nails, gastrointestinal tissues, oral tissues, teeth, taste buds, urogénital tissues, rénal tissues, reproductive tissues, lymphatic tissues, immune system tissues/elements and such related appendages and protein cellular éléments.
This invention contemplâtes the direct delivery of supported LGR expressing épithélial stem cells by application, transplantation, implantation, directed seeding, directed migration, directed tracking, in setting, iaminating and/or injection of the cellular element to alt®r mammalian tissue(s) in therapeutics, devices, biologics, drugs and bio-engineering.
Définitions
In this detailed description, référencés to one embodiment, an embodiment”, or “in embodiments” mean that the feature being referred to is included in at least one embodiment of the invention. Moreover, separate references to one embodiment, an embodiment, or “embodiments” do not 30 necessarily refer to the same embodiment; however, neither are such embodiments mutually exclusive, unless so stated, and except as will be readiiy apparent to those skïlled in the art. Thus, the invention can include any variety of combinations and/or intégrations of the embodiments described herein. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
As used herein, the singular forms, “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.
It will be further understood that the root terms “include” and/or “hâve”, when used in this spécification, specify the presence of stated features, steps, operations, éléments, and/or components, but do not preclude the presence or addition of at least one other feature, step, operation, element, component, and/or groups thereof.
As used herein Bone means the hard connective tissue consisting of cells embedded in a matrix of mineralized ground substance and collagen fibers. The fibers are impregnated with inorganic components, including crystals of calcium phosphate, such that using X-ray defraction, they are seen to be organized in a hydroxyapatite pattern (calcium phosphate is 85% by weight) as well as calcium carbonate (10%), and magnésium; by weight, bone is composed of 65-75% inorganic and 25-35% organic material; a portion of osseous tissue of definite shape and size, forming a part of the animal skeleton; in humans there are approximately 200 distinct bones in the skeleton, not including the auditory ossicles of the tympanic cavity or the sesamoid bones other than the two patellae. A bone is enveloped by a fibrous membrane, periosteum that covers the bone's entire surface except for the articular cartilage. Beneath the periosteum is a dense layer, compact bone, and beneath that a cancellous layer, spongy bone. The core of a long bone is filled with marrow.
As used herein, the terms “comprises,” “comprising,” “includes,” “including,” “has,” “having” or any other variation thereof, are intended to cover a non-exclusive inclusion. For example, a process, method, article, or apparatus that comprises a list of features is not necessarily limited only to those features but may include other features not expressly listed or inhérent to such process, method, article, or apparatus.
As used herein Epithelium means the cellular layer covering ail free surfaces, cutaneous, mucous, and serous, including the glands and other structures derived therefrom.
As used herein GMP means good manufacturing practices.
As used herein Integument means the enveloping membrane of the body; includes, in addition to the epidermis and dermis, ail the dérivatives of the epidermis, hairs, nails, sudoriferous and sebaceous glands, and mammary glands, as well as the subcutaneous tissue.
As used herein LGR4 means Leucine-Rich Repeat Containing G Protein-Coupled Receptor 4, G protein-coupled receptors (GPCRs) that play key rôles in a variety of physiologie functions. Members of the leucine-rich GPCR (LGR) family, such as GPR48, hâve multiple N-terminal leucine-rich repeats (LRRs) and a 7-transmembrane domain. LGR4 (Leucine-Rich Repeat Containing G Protein-Coupled Receptor 4) is a Protein Coding gene. Diseases associated with LGR4 include bone minerai density, low. Among its related pathways are Wnt signaling pathway (KEGG). GO annotations related to this gene include G-protein coupled receptor activity and transmembrane signaling receptor activity. An important paralog of this gene is LGR6. Receptor for R-spondins that potentiates the canonical Wnt signaling pathway and is involved in the formation of various organs. Upon binding to R-spondins (RSPO1, RSPO2, RSPO3 or RSPO4), associâtes with phosphorylated LRP6 and frizzled receptors that are activated by extracellular Wnt receptors, triggering the canonical Wnt signaling pathway to increase expression of target genes.
In contrast to classical G-protein coupled receptors, LGR4 does not activate heterotrimeric G-proteins to transduce the signal. Its function as activator of the Wnt signaling pathway is required for the development of various organs, including liver, kidney, intestine, bone, reproductive tract and eye. LGR4 may also act as a receptor for norrin (NDP) and is required during spermatogenesis to activate the Wnt signaling pathway in perïtubular myoid cells. Likewise, LGR4 is required for the maintenance of intestinal stem celte and Paneth cell différentiation in postnatal intestinal crypts. LGR4 also acts as a regulator of bone formation and remodeling in addition to being involved 5 in kidney development; required for maintaining the ureteric bud in an undifferentiated State. LGR4 is involved in the development of the anterior segment of the eye, required during erythropoiesis and also acts as a négative regulator of innate immunity by inhibiting TLR2/TLR4 associated pattern récognition and pro-inflammatory cytokine production. LGR plays an io important rôle in regulating the circadian rhythms of plasma lipids, partially through regulating the rhythmic expression of MTTP (By similarity). Commonly known aliases for LGR4 include: GPR48; G Protein-Coupled Receptor 48; BNMD17; Leucine-Rich Repeat-Containing G Protein-Coupled Receptor 4; Leucine-Rich Repeat-Containing G-Protein Coupled Receptor 4;
and G-Protein Coupled Receptor 48. External Database Identifiera for LGR4 include: HGNC: 13299 Entrez Gene: 55366 Ensembl: ENSG00000205213 OMIM: 606666 and UniProtKB: Q9BXB.
As used herein LGR5 means Leucine-Rich Repeat Containing G ProteinCoupled Receptor 5, a Protein Coding gene. Among its related pathways are 20 Wnt signaling pathway (KEGG). GO annotations related to this gene include G-protein coupled receptor activity and transmembrane signaling receptor activity. An important paraiog of this gene is LGR6. The LGR5 Receptor is for R-spondins that potentiates the canonical Wnt signaling pathway and acts as a stem cell marker of the intestinal epithelium and the hair follicle. Upon binding to R-spondins (RSPO1, RSPO2, RSPO3 or RSPO4), associâtes with phosphorylated LRP6 and frizzled receptors that are activated by extracellular Wnt receptors, triggering the canonical Wnt signaling pathway to increase expression of target genes. In contrast to classicai G-protein coupled receptors, LGR5 does not activate heterotrimeric G-proteins to transduce the signal. Involved in the development and/or maintenance of the adult intestinal stem cells during postembryonic development. Commonly known aliases for LGR5 include: G-Protein Coupled Receptor HG38; GProtein Coupled Receptor 49; G-Protein Coupled Receptor 67; GPR67;
-----................................ GPR49and Leucine-Rich Repeat-Containing G-Protein Coupled Receptor 5.External Database Identifiera for LGR5 include HGNC: 4504 Entrez Gene: 8549 Ensembl: ENSG00000139292 OMIM: 606667 and UniProtKB: 075473.
As used herein LGR6 means Leucine-Rich Repeat Containing G Proteins Coupled Receptor 6 which is a Protein Coding gene a gene that encodes a member of the leucine-rich repeat-containing subgroup of the G proteincoupled 7-transmembrane protein superfamily. The encoded protein is a glycoprotein hormone receptor with a large N-terminal extracellular domain that contains leucine-rich repeats important for the formation of a horseshoe10 shaped interaction motif for ligand binding. Alternative splicing of this gene results in multiple transcript variants. Diseases associated with LGR6 include myxedema and ovarian cystadenoma. Among its related pathways are Wnt signaling pathway (KEGG) and GPCRs, Other annotations related to this gene include G-protein coupled receptor activity and transmembrane ta signaling receptor activity. An important paralog of this gene is TSHR.
Receptor for R-spondins that potentiates the canonical Wnt signaling pathway and acts as a marker of multipotent stem cells in the epidermis. Upon binding to R-spondins (RSPO1, RSPO2, RSPO3 or RSPO4), associâtes with phosphorylated LRP6 and frizzled receptors that are 20 activated by extracellular Wnt receptors, triggering the canonical Wnt signaling pathway to increase expression of target genes. In contrast to classical G-protein coupled receptors, LGR6 does not activate heterotrimeric G-proteins to transduce the signal and can act as a tumor suppressor.
Common aliases for LGR6 include: Gonadotropin Receptor; VTS20631 and 25 GPCR. External Database identifiera for LGR6 include HGNC: 19719 Entrez
Gene: 59352 Ensembl: ENSG00000133067 OMIM: 606653 and UniProtKB: Q9HBX8.
As used herein Mesenchyme means an aggregation of mesenchymal cells. Primordial embryonic connective tissue consisting of mesenchymal 30 cells, usually stellate in form, supported in snter-laminar jeily.
As used herein Muscle means the primary tissue, consisting predominantly of highly specialized contractile cells, which may be classified as skeletal muscle, cardiac muscle, or smooth muscle; microscopically, the
——...............|atter is lacking in transverse striations characteristic of the other two types; one of the contractile organs of the body by which movements of the various organs and parts are effected; typical muscle is a mass of musculus fibers (venter or belly), attached at each extremity, by means of a tendon, to a bone or other structure; the more proximal or more fixed attachment is called the orïgin (q.v.), the more distal or more movable attachment is the insertion (q.v.); the narrowing part of the belly that is attached to the tendon of origin is called the caput or head.
As used herein Neural is intended to include any structure composed of ίο nerve cells or their processes, or that on further development will evolve into nerve cells. Referring to the dorsal side of the vertébral bodies or their precursors, where the spinal cord is located, as opposed to hemal.
As used herein, and unless expressly stated to the contrary, or” refers to an inclusive-or and not to an exclusive-or. For example, a condition A or B is satisfied by any one of the following: A is true (or présent) and B is false (or not présent), A is false (or not présent) and B is true (or présent), and both A and B are true (or présent).
The meaning of Particle herein connotes the largest domain of which is ten micron or less and includes, but is not limited to, nanoparticles, an association of macromolecules, a micelle, a cell ghost, a dendrimer, and the like that can serve as a suitabie anchorfor a cell micro-aggregate.
As used herein Polarity means the tendency of a cell, tissue(s) and/or organism to develop differentially along an axis.
As used herein Puise Rescue Media (PRM) is a formulation of a cell sustaining media mixture including Keratinocyte-SFM (1X), an antibioticantimycotic selected from the group consisting of penicillin, streptomycin, and amphotericin B, and fibrinogen where the Keratinocyte-SFM is composed of a mixture of épithélial cells and kératinocytes. The reagents are utilized in order to stabilize the primary tissues and reduce the viability of micro- organisms during transport and processing.
As used herein Skin means the membranous protective covering of the body, consisting of the epidermis and dermis (corium).
As used herein Stem cell means any precursor cell; a cell with daughter
cells that may differentiate into other cell types; a cell capable of maintaining its own number while exporting progeny to one or more cell lineages.
As used herein substantially, generally, and other words of degree are relative modifiers intended to indicate permissible variation from the characteristic so modified. It is not intended to be limited to the absolute value or characteristic which it modifies but rather possessing more of the physical or functional characteristic than its opposite, and preferably, approaching or approximating such a physical or functional characteristic.
As used herein Tissue means a collection of similar cells and the intercellular substances surrounding them. There are four basic kinds of tissue in the body: epithelium; connective tissues including adipose tissue, blood, bone, and cartilage; muscle tissue; and nerve tissue. The rind, capsule, or covering of any body or part.
In the following description, reference is made to the accompanying drawings which are provided for illustration purposes. The following illustrated embodiment is described in sufficient detail to enable those skilled in the art to practice the invention. It is to be understood that other embodiments may be utilized and that structural changes based on presently known structural and/or functional équivalents may be made without departing from the scope of the invention.
Brief Description of the Drawings
Figure 1A depicts an example of location of LGR expressing cells of cutaneous origin.
Figure 1B is a Fluorescent Activated Cell Sorting graph.
Figure 1C are photographs of a spectrum of various acellular supports contemplated for use in connection with the invention.
Figure 2A is photograph of a gross cellular construct/ de-cellularized collagen scaffold usable for seeding.
Figure 2B are immunofluorescent photomicrographs of a collagen construct following seeding with aggregates of partially digested cells.
Figure 3A - 3F présent various images by different techniques of an array of different LGR6+ épithélial stem cell seeded substrates.
Figure 4A depicts a time lapse in vivo healing progression of Controls and an example of an LGR seeded matrix. Figure 4B is a graphical expression of Cytokeratin-17 transcript expression at day ten. Figures 4C-E depict Controls and a matrix seeded with LGR ESC by bioluminescent imaging and scanning électron microscopy.
Figures 5A-E depict an example of a construct with LGR ESCs and stromal vascular fraction cellular isolate populations showing initial form of polarization accompanied by a graphie comparison.
Figures 6A-B depict an example of a construct containing LGR cells 10 with and without stromal vascular fraction cellular entities and the relative production of growth factors.
Figures 7A-H illustrate third degree wound bed induction and vérification ofthe élimination ofthe LGR stem cell follicular bulge and adnexal structures.
Figures 8A-Q depict time progression of a wound/injury/void with
DEFA5 as it relates to bacterial adhesion.
Figures 9A and B are comparative photographs of DEFA5 expressing cellular entities within a wound bed as it relates to augmented healing, tissue and appendage régénération and subséquent hair growth in treated burn wounds devoid of adnexal structures.
Figures 10A-L illustrate the quantification of wound bed healing kinetics and LGR5 and LGR6 stem cell migration into burn tissue following treatment with topical focal agents.
Figures 11A and B illustrate RT-PCR quantification and gene heat mapping comparison of wound/injury/tissue voids treated with DEFA5 to SDZ as it relates to augmentation of pro-healing pathways.
Figures 12A-I illustrate LGR6 expression of cells ofthe hair follicle and fluorescent activated cell sorting of co-expressing LGR6+, CD34+ CD73+ GFP labeled cells for culture expansion.
Figures 13A-D are photomicrographs by confocal microscopy and bioluminescence of a functional singularity unit (aFSU) at the time initial seeding and 1 day later.
Figures 13E is a photomicrograph of a collagen scaffold.
Figures14A-E depict an example of location LGR cellular varieties as it relates to location, phenotype, interface and polarity within a cutaneous tissue. Isolation and culture of the LGR6+ ESC from the follicular bulge.
Figures 15A-E provides an example of LGR expressing cellular foci as it relates to a method of delivery through placement around and/or within wound/injury/tissue void.
Figures 16A-D depict an example of LGR containing stem cell as it relates to delivery into and around wounds via a deliverable vector and subséquent healing, régénération of tissues and supporting structures.
Figures 1 7A-D show LGR6+ épithélial stem cell migration and différentiation within full-thickness wound beds 10 days after transplantation.
Figure 18 provides an RT-PCR quantification and inset gene heat mapping comparison of a wound/injury/tissue void with the LGR expressing 15 cellular foci.
Figure 19 depicts an example of said LGR expressing cellular foci as it relates to delivery into and/or around wound/injury/tissue void and augmentation of wound healing factors.
Figures 2 0A-F illustrate an example of LGR expressing cellular foci as it relates to the régénération of bone tissues. Isolated LGR foci can be seeded bone and remain viable.
Detailed Description of the Drawings
Figures 1A-C Example of flow cytometry of cell populations that exist around a hair follicle and scaffolds that such cells readily adhéré to when seeded. More specifically, Figure 1A depicts an example of location of said LGR expressing cells of cutaneous origin. Immunofluorescent confocal microscopy at 40x magnification depicts the follicular bulge (white arrow), LGR6+ (Green), DNA (Blue). Figure 1B is a fluorescent activated cell sorting graph with gâte analysis indicating exemplary cellular markers. Figure 1C depicts an array of cells types can be used to seed a spectrum of acellular matrices/substrates/scaffolds/materials according to the invention.
Figure 2A is a photographie représentation of an example of a gross construct without micro-aggregate multi-cellular functional units containing LGR expressing stem cell foci in accordance with the invention. Figure 2B depicts the construct following seeding of substrate with aggregates of 5 partially digested cells.
Figure 3A, in columnar format, is an image sériés by differential interférence contrast (DIC) confocal microscopy of LGR seeded substrates from different sources. Figure 3B is a corresponding column by immunofluorescent confocal microscopy at 20x magnification of LGR6+ ESC 10 seeded matrices of respective constructs containing LGR expressing cells.
The inset white boxes represent focal zoom régions indicated in the column Figure 3D while Figure 3C is a column depicting the Digital merge of the respective image of Figure 3A (DIC) and the immunofluorescent of Figure 3B indicating matrix contour and boundaries. The columns of Figures 3E and 3F 15 respectively represent the bioluminescence measured in radiant efficiency of an acellular matrix control and a corresponding LGR6+ ESC seeded matrix at 72 hours post-seeding.
Figure 4A-E depict examples of said LGR containing construct placed into living mammalian system. Placement of an LGR6+ GFP ESC Seeded 20 Matrix Augmente Healing Haïr Follicle Growth. Figure 4A is a 3x3 matrix of photomicrographs of 3mm full human de-cellularized dermis thickness burn wound beds at days 5, 8 and 10 containing no matrix (burn control), matrix (matrix control) and LGR6+ GFP ESC. Figure 4B graphically depicts the relative expression of Cytokeratin-17 transcript expression at day 10 of the 25 wound beds depicted in Figure 4A. The percent wound bed healed was determined using quantification analysis of wound bed healing rates as a percent area function within the ImageJ NCBI application. Wound control contains burn wound bed only. Matrix control contains matrix only and LGR6+ GFP contains ADM seeded with LGR6+ GFP ESCs.
Figure 4C is a photomicrograph of in vivo bioluminescent imaging in murine full thickness burn wound beds at day 5. Figure 4D are micrographs of human dermis at 100x of the Controls and LGR6+ GFP containing dermis at 12 hours and 72 hours and after seeding with ESCs. The white arrow indicates the presence of a dermal pore Figure 4E provides images of the Controls and the construct of the invention containing human dermis seeded with ESCs with a silicone protective overlay to prevent desiccation. The LGR6+ GFP matrix image includes duplicate small black arrows that indicate 5 nascent hair patches from the full thickness Nu/Nu murine wound bed.
Figures 5A-E depict an example of said construct the effect of addition of Stromal vascular fraction (SVF) to LGR6+ ESC Seeded Matrices in promoting tissue polarization and a dual compartment skin-like System.
Figure 5A is confocal 20x imaging of a 5x105 RFP expressing stromal vascular 10 fraction cellular isolate population 24 hours after being seeded on to a représentative Adrenomedullin (ADM) (such as that available from Integra LifeSciences Corporation under the name INTEGRA®). Figure 5B is a confocal 20x image of a 5x105 GFP expressing LGR6+ cellular isolate population 24 hours after being seeded on to a représentative ADM (INTEGRA®). Figure 5C depicts confocal 20x imaging of a dual seeded représentative ADM (INTEGRA®) with 5x105 RFP expressing SVF and 5x105 GFP expressing LGR6+ isolate populations 24 hours after being co-seeded in culture. Figure 5D is of a co-seeded matrix containing 5x105 RFP expressing SVFRFPand 5x105 GFP expressing LGR6+ following 5 days of growth in culture. The dotted parallel lines indicate épithélial LGR6+GFP lineage accumulating at the edge of the ADM substrate. The small bracket and large bracket indicate the relative locations of the two compartments in corrélation with LGR6+GFP and SVFRFP abundance. The arrowed “U” shaped solid line indicates a région containing a pre-seeded pore induced by a 32 gauge stérile needle. Figure 5E is a graphical représentation ofthe prolifération kinetics of a collagen substrate co-seeded with green LGR expressing cells and red SVF expressing cells.
Figures 6A and 6B depict an example of a construct containing LGR cells with and without supportive cellular entitîes and the relative production of 30 growth factors. Corrélative Expression Profiles of Pro-angiogenic Transcripts and Protein Analytes from LGR6+GFP ESC and SVFRFP Enriched Scaffolding Culture Constructs. Figure 6A graphs relative fold transcript expression (AACT) of indicated gene element from total RNA: LGR6+GFP ESC (black bar), SVFRFP (grey bar), and co-cultured LGR6+GFP ESC + SVFRFP (white box) on respective scaffold substrate. Significance above the x-axis (LGR6 + SVF) indicates the inter-comparison co-cultured LGR6+GFP ESC + SVFRFP expression vs. singular LGR6+GFP ESC and SVFRFP expression on indicated scaffolding. Ex. Average FGF-2 gene expression for co-cultured matrices was 5 higher than the average expression of both singular Systems (Scaffold+ LGR6 or scaffold +SVF) except for co-cultured INTEGRA® (INTEGRA®+ LGR6+ SVF). Significance below the x-axis (LGR6) or (SVF) indicates the intracomparison of substrates, while the cellular entity remains constant. Ex. VEGF-A gene expression for INTEGRA®+ LGR6+GFP ESC only vs.
DERMAMATRIX®+ LGR6+GFP ESC only was nonsignificant (NS). Figure 6B graphically represents the relative densitométrie unit (RDU) of indicated protein analyte from total protein isolâtes: LGR6+GFP ESC (black bar), SVFRFP (grey bar), and co-cultured LGR6+GFP ESC + SVFRFP (white box) on respective scaffold substrate. (*) indicates (p-value < 0.05), assays completed in triplicates, GAPDH housekeeping control.
Figures 7A-H illustrate a wound/injury/void receiving therapy example of enhanced LGR cell migration, prolifération and viability into a wound namely a third degree wound bed induction and vérification ofthe élimination ofthe LGR stem cell follicular bulge and adnexal structures. Figure 7A depicts 20 a wound bed template marks of 3mm diameter. Figure 7B depicts the wound bed structure at day 0 (the white scale bar being 1 mm). Figure 7C illustrâtes an example of a 2x3 3mm wound bed grid. Figure 7D shows topical application ofthe re-suspended peptide at the wound site. Figure 7E is a photomicrograph of H&E stain of non-bumed, intact Integument/skin with hair 25 follicle and adnexal structures. The arrow indicates the location of the magnified follicle (inset image) where the white scale bar is 500pm. Figure 7F is an H&E stain of dorsal murine skin following high température cautery depicting removal of epidermal, dermal and hypodermal tissues including the follicular bulge. Figure 7G is DAPI/DNA stain (4’,6-diamidino-2-phenylindole) of non-burned, intact skin with hair follicle and adnexal structures. The arrow indicates the magnified follicle with co-labeling of immunofluorescent LGR5 and LGR6 antibodies green and red respectively (inset image). Figure 7H DAPI/DNA stain of dorsal murine skin following high température cautery depicting removal of epidermal, dermal, and — — hypodermal tissues including the follicular bulge where the white scale bar is 100 — pm.
Figures 8A-Q depict a wound/injury/void with LGR as it relates to antimicrobial behavior over five and ten day time periods. Using 16S rRNA fluorescent oligonucleotide probes, in-situ hybridization indicates the presence of bacterial adhesion at the third degree burn wound bed. Figure 8A présents DNA/DAPI labeling of a 3rd degree burn wound bed at day five post burn induction treated daiiy with SDZ. In Figure 8B 5’- Cy3-EUB338 labeied 16s rRNA of 3rd degree burn wound bed bacterial organisme (yeîiow grains) at day io five post burn induction treated daiiy with SDZ are depicted. Figure 8C is a digitaily merged image of Figures 8A and 8B. Figure 8D corresponds to Figure 8Α except at day ten with DNA/DAPI labeiing of 3rd degree burn wound bed treated daiiy with SDZ. Correspondingly, Figure 8E is a photomicrograph of the 5'- Cy3-EUB338 labeied 16s rRNA of 3rd degree burn wound bed bacterial organisme (yellow grains) at day ten post burn induction treated daiiy with SDZ. Figure 8F is a merged image of Figures 8D and E. Figures 8G-8L are images corresponding respectively to the five and ten post burn periods of Figures 8A-F but subject to daiiy treatment using Defensin, alpha 5 (DEFA5) rather than SDZ. The arrow in H represents the interface of tissue with overlying fibrinous material where less bacteria is observed in the setting of DEFA5 treatment.
Figure 8M with inset 8N demonstrate quantification of white pixel intensity of Cy3 fluorescence grayscale converted image of a wound bed treated with SDZ and containing more 16s rRNA labeling per unit area. Figure 80 and inset 8P correspondingly show quantification of white pixel intensity of Cy3 fluorescence grayscale converted image of (inset image p.) a wound bed treated with DEFA5 and containing a reduced 16s rRNA labeiing per unit area. The inset graph depicts averaged white pixel intensity of 16s rRNA expressed in both SDZ and DEFA5 treated burn wound beds at day five using grayscale imaging software. Finally, Figure 8Q is a graph to illustrate averaged red channel fluorescence of
16s rRNA expressed in both SDZ and DEFA5 treated burn wound beds at day five. The white arrow in Figure 8H indicates potentiel film in DEFA5 treated wound beds and the black arrow in Figure 8M indicates whitepixel intensity. Scale bar ΙΟΟμπη. (*) indicates p-value <0.05.
....................................... Figures 9A and B-are a sériés of time progression photographe that---—represents an example of LGR expressîng cellular entities within wound as it relates to augmented healing, tissue and appendage régénération and subséquent haïr growth, wound healing kinetics and nascent haïr growth in treated burn wounds devoid of adnexal structures. The photographie sériés comprising Figure 9A are gross imaging using a Leica Wild M680 surgical microscope to image healing of 3rd degree burn wound beds over 10 days white being treated with indicated agents MQH2O, DEFA5, DEFB1, SDZ. The white scale bar represents 1 mm. The second photographie sériés of Figure 9B again io comprises gross imaging using a Leica Wild M680 to track nascent hair growth of 3rd degree burn wound beds over 16 days in a side by side comparison of DEFA5 vs. control treated wound beds. The white arrows indicate the growth of new hair. Again, the scale bar is 1mm.
Figures 10A-L comprise an example of said LGR expressing cellular entities within wound/injury/ tissue void as it relates to augmented healing, propagation of said entities. The Graphe comprising Figures 10K and 10L provide evidence of quantification of wound bed healing kinetics and LGR5 and LGR6 stem cell migration into burn tissue following treatment with topical focal agents. Briefly, these tests were used to confirm the quantitative confocal microscopie intensity patterns from imaging LGR5 and LGR6, and based on reverse-transcriptase polymerase chain reaction on burn wound tissues. As represented in the graphs, averaged LGR5 and LGR6 mRNA expression within human alpha defensin 5 wound beds was found to be 95.8 ± 10.6 and 259.2 ± 20.2, respectively, compared with undetectable levels of LGR5 and LGR6 in sulfadiazine-treated wounds at day 5 (Fig. 4, right). The magnitudes of these fold-level comparisons within human alpha defensin 5-treated tissues and those specïmens treated with sulfadiazine suggest that it is the absolute presence or void of cells expressîng LGR5 and LGR6 migrating into the wound that defines the fold values.
so Turing to the spécifie figures, Figure 10A présents photographs of a wound area with a white scale bar representing 1mm and the wound area calculation in black. Figure 10B graphically displays the averaged wound healing rate expressed as percent % of wound area remaining over 10 day ,-.....:............... period of indicated topical focal agent application. The asterisk (=) represents a— p-value <0.05. Figures 10C-J are LGR5 and LGR6 immunofluorescent antibody labeling of a DEFA5 treated wound bed at day 5 where Figure 10C is DNA/DAPI/Blue, Figure 10D is LGR5/FITC/Green Figure 10Eis
LGR6/TRITC/Red and Figure 10F is a merger of 10C-10E. Figures 10G-I are corresponding LGR5 and LGR6 immunofluorescent antibody labeling of SDZ (sulfadiazine) treated wound bed at day 5 (DNA/DAPI/Blue, LGR5/FITC/Green and LGR6/TRlTC/Red). Figure 10J is a merged image of 10G-10I and includes an inset representing averaged LGR5 and LGR6 expression using Green and io Red fluorescent intensity per wound bed at day 5. Th© comparative values obtained from Reverse Transcriptase PCR quantification of the fold increase in RNA extracted from replicate wound beds treated with DEFA5 and SDZ is set out The white scale bar 50pm and again, the asterisk (*) represents a p-value <0.05.
Figures 11A and B illustrate a wound/injury/tissue void with th© LGR expressing cellular entities placed within wound as it relates to augmentation of pro-heaiing pathways. The figures respectively represent RT-PCR quantification and gene heat mapping comparison of wound beds treated with DEFA5 to SDZ. These figures show the rôle of human aipha defensin 5 versus sulfadiazine in augmenting key transcript expression within the wound. The results show that several gene subsets are signifîcantly up-reguiated within the wound beds receiving human alpha defensin 5 when compared with sulfadiazine therapy and that certain Wnt pathway gene subsets are significantiy up-regulated in response of the LGR stem cell system to Wnt ligands in both the gut and skin.
Figure 11A présents an Averaged Wound Healing RT2-PCR Array pathway heat map and corresponding gene map with fold régulation for wound beds comparing DEFA5 to SDZ treated Systems. Figure 11B présents an Averaged Wnt RT2-PCR Array healing pathway heat map and corresponding gene map with fold régulation for wound beds comparing DEFA5 to SDZ treated
Systems. The cotors of the heat maps are indicated as red, more expressed in DEFA5 treated bums to green more expressed in SDZ treated burns.
Figures 12A-I represent an example of a micro-aggregate muiticellular unit containing LGR expressing stem cell foci as it relates to location, population
................. -.....: identity and wound healing capacity. Using a simple ex vivo wound healing assay and fluorescence-activated cell sorting, LGR6+, CD34+, and CD73+ C57BL/6(UBC-GFP) murine celte were isolated for cell culture expansion.
Figure 12A depicts LGR6 fluorescent antibody (green) expression of cells on the hair follicle following partial epidermal 10 unit/pL dispase digestion. (Worthington Biochemical Corp., Lakewood, NJ.) digestion for 30 minutes at 37°C on a slow rocker. Figure 12B is of LGR6+ cells expressing additional CD34 and CD73 markers (the arrow indicates population isolated comprising approximately 1 to 3 percent of ail cells). Figures 12C-H are eFluor450 expression histograms of an io in vitro wound assay respectively showing periodic intrinsic GFP expression from C57BL/6(UBC-GFP) murine cells, CD34+ PE/Cy7 expression, LGR6+ APC expression and CD73+. The dotted lines indicates the distance of séparation at 0, 6, and 12 hours following disruption of the cell layer and the scaie bar = 50 pm. The graph of Figure 121 sets out the averaged réduction in the distance line is over time expressed as a percentage of initial distance foliowing fluorescence sorting where the asterisk (*) represents a p-value <0.05.
Figures 13A-D are photomicrographs by confocal microscopy and bioluminescence of an activated functional singularity unit (aFSU) at the time initial seeding and 1 day later showing an example of a micro-aggregate multicellular unit containing LGR expressing stem cell foci while undergoing initial propagation on a collagen matrix, Figure 13 E.
Figures14A-E depict an example of location LGR cellular varieties as it relates to location, phenotype, interface and polarity within a cutaneous tissue. Figure 14A shows by Immunofluorescence stainïng, localized régions of LGR6 (Green/fluorescein isothiocyanate (FITC)) and LGR5 (Red/tetramethyl rhodamine isothiocyanate (TRITC)) expression. The scale bar is for 20pm. Figure 14B shows fluorescence-activated cell sorting isolation of the LGR6+GFP épithélial stem cells from C57BU6(UBCGFP) murine skin with the final sort gâte using LGR6+, CD34 and CD73 on the left and individual histograms depicting so cellular GFP expression and correlating antibody-conjugate labels: CD73/PE-7, LGR6/Cy5, CD34/eF!our450 on the right. Figure 14C shows différentiel interférence contrast image of LGR6*GFP épithélial stem cells piated following fluorescence-activated cell sorting isolation. Figure 14D depicts intrinsic GFP expression of the LGR6+GFP épithélial stem cells and Figure 14E is a merged image of Figures 14C and 14 D. The scale bar represents 20pm.
Figures 15A-E provide an example of LGR expressing cellular foci as it relates to a method of delivery through placement around and/or within wound/injury/tissue void. The three images of Figure 15A depict, respectively, an initial burn template; a full thickness burn on the dorsum on Nu/Nu mouse; and delivery of HYDROGEL® containing 105 LGR6+GFP épithélial stem cells at the base of the wound bed. The scale bar for Figure 15A is 1mm. Figure 15B is an immunofluorescece image of the injection pocket DNA/DAPI-BLUE at Day 0 Figure 10 15B is an immunofluorescece image of anti-LGR6/TRITC antibody labeling and
Figure 15C the same for LGR6+GFP épithélial stem cells. Figure 15 E is a merged image of Figures 15B-D and has a scale barof 20pm. Figures 15A-E show full thickness burn wound bed induction and validation of LGR6+ stem cell engraftment into subséquent soft tissue defect.
Figures 16A-D depict an example of LGR containing stem cell focus as it relates to delivery into and around wounds via a deliverable vector and subséquent healing, régénération of tissues and supporting structures including but not limited blood vessel angiogenesis and/or angiogenesis. Wound healing progression following LGR6+ épithélial stem cells transplantation into full thickness wounds.
The progression of wound healing is depicted following the injection of HYDROGEL® from BD Biosciences, San José, Calif. (control) in Figure 16A compared with Figure 16B, LGR6+GFP épithélial stem cells seeded HYDROGEL® over 15 days. The scale bar is 1mm. In Figure 16C, showing the implant pocket after day 15, the white arrow indicates presence of a remaining LGR6+GFP épithélial 25 stem cells population located within healing wound bed. In Figure 16D, the black arrow indicates the location of the burn wound base free of LGR6+GFP épithélial stem cells.
Figures 17A-D depicts an example of LGR containing stem cell focus following delivery into and/or around wound with subséquent healing and 30 régénération of tissues and related appendages such as but not limited to hair follicle and related supportive structures. Figure 17A is a four panel matrix of confocal images of immunofluorescent labeled tissue specimen at day 10 following transplantation of LGR6+ épithélial stem cells migration into the wound
-—— —...................bed 10 days. The images comprising Figure 17A include DNA/DAPI-BLUE; anti~LGR6/TRITC; GFP expression of LGR6*GFP ESC.
Figure 17B is a différentiel interférence contrast image merge of ail channels. The Red arrow désignâtes régions of nascent follicle development.
(See also the upper inset image). The dotted line shows épithélial polarization overlying nascent hair follicles whiie the white arrow indicates the location of the graft injection pocket (See also the magnification thereof in the lower inset image for an image ofthe initial injection pocket cellular population. The inset graph of Figure 17B represents comparative KRT17/ cytokeratin 17 gene expression to within the indicated wound beds of the control and LGR6++GFP treatment.
Referring to Figure 17C, the three images are of a Transplant dôme used to cover hair follicle study population burn wound beds, an LGR6++GFP ESC treated wound bed at day 10 (solid arrow) with nascent hair follicles (clear arrow) follicle cyst formation and a control wound bed at day 10. The graph comprising
Figure 17D quantifies the Day 10 wound bed resulting from RT-PCR indicating relative gene fold expression of WNT ligands. The positive numbers indicated higher expression in LGR6+GFP épithélial stem cells wound beds while the négative numbers indicate higher expression in control wound beds.
Figure 18 provides an RT-PCR quantification and inset gene heat mapping comparison of a wound/injury/tissue void with the LGR expressing cellular foci as it relates to delivery into and/or around wound/injury/tissue void as it reiates to augmentation of pro-healing pathways and comparative gene expression of wounds receiving LGR6+ épithélial stem cells against a control. The graphs illustrate the relative fold expression of genes for angiogenesis, wound healing and epidermal growth factor. Corrélative graphicai représentation of data comparing wound beds receiving LGR6+ épithélial stem cells and control therapy. As to the inset heat maps the color red indicates greater expression within the LGR6+ épithélial stem cell wound bed whiie the color green indicates greater expression within the control wound bed. In the bar graphs, positive numbers indicated higher expression in LGR6+GFP épithélial stem cell wound beds and négative numbers indicate higher expression in control wound beds. The NCBI Unigene term is indicated at the top of each quantitative column and the asterisk (*) P-value désignâtes <0.05 significance.
—......................................................-Figure 19 graphicaily présents the relative protein densitometry of an example of —
LGR expressing cellular foci as it relates to delivery into and/or around wound/injury/tissue void and augmentation of wound healing factors.
Comparative angiogenesis analyte expression of wounds receiving LGR6+
ESCs Proteomic array comparing common proteins which regulate and augmented angiogenesis. The grey columns indicate control wounds and the black columns indicated those wounds that received the LGR6+GFP ESC. The inset image shows example proteome array membranes following development with HRP chemi-luminesce. Brighter colors indicate higher levels of protein expression.
Figures 20A-F illustrate an exampie of LGR expressing cellular foci as it relates to the régénération of bone tissues. Isolated LGR foci can be seeded bone and remain viable. Figure 20A is a gross bone image of harvested bone for culture. Figure 20B is a D1C image of bone containing LGR GFP 7 days following seeding. Figure 20C is a 488 nm Green laser confocal image of bone containing LGRe*6^ 7 days following seeding. It is notable that the LGR foci can undergo osteo-induction in-vitro. Figure 20D depicts LGR foci following 1 week of osteo-induction with supplémentai media. Figure 20E is an Alizarin red stain of the LGR foci following osteo-induction which can undergo osteo20 induction in-vitro and up regulate key ostéogénie genes. Finally, Figure 20F is RT-PCR data showing relative fold gene expression where the grey columns represent (control) non-osteo induced LGR and the black columns represent those LGR which received osteo-induction media following 7 days of culture. GAPDH was used as reference standard housekeeping gene.
EXEMPLARY PROTOCOL
The following is a sériés of examples providing an illustrative protocol sequence for practice of an embodiment of the invention.
Prior to génération of the minimally polarized functional units in accordance with the invention, a gelatinous support such as an exemplary three dimensional collagen scaffold can be generated by well-known processes as follows:
i. Slowly adding 1 part of chilled 10X PBS of 10X culture media to 8 parts of chilled collagen-based solution with gentle swirling. Adding ECM and viability proteins to the suspension;
ii. Adjusting the pH of mixture to 7.2-7.6 using stérile 0.1M NaOH and monitoring the pH adjustment carefully;
iii. Adjusting the final volume to a total of 10 parts with stérile molecular grade water;
iv. Maintaining température of mixture at 2 - 10°C to prevent gélation, v. Forming a gel by warming to 37°C for approximately 90 to 120 minutes;
vi. Perforating the scaffold with a stérile micro-needle press (the scaffold can undergo freeze drying process if needed for storage).
It is also recommended that an additional material referred to as Puise Rescue Media (PRM) be produced and be available prior to commencement of the LGR aggregate extraction procedures.
The PRM, in this embodiment which is direct to humans, is a cell sustaining, serum-free, media mixture Keratinocyte-SFM containing Lglutamine supplied with separately packaged prequalified human recombinant Epidermal Growth Factor 1-53 (EGF 1-53) and Bovine Pituitary Extract (BPE) sold as Keratinocyte-SFM (1X) from Thermo Fisher Scientific to which the antibiotic-antimycotic agents penicillin, streptomycin, and amphotericin B are added along with a GMP- fibrinogen: human. The agent used in one embodiment is GIBCO® Antibiotic-Antimycotic from Thermo Fisher Scientific, a solution containing 10,000 units/mL of penicillin, 10,000 pg/mL of streptomycin, and 25 pg/mL of FUNGIZONE® Antimycotic.
Because the PRM is used to transport human tissues, the supplémentai reagents are utilized to stabilize the primary tissues and reduce the viability of micro-organisms during transport and processing.
The following relates specifically to the génération and préservation of LGR expressing épithélial containing stem cell micro-aggregate functional units in accordance with an embodiment ofthe invention.
Example 1
Example 1 concerns a method for extraction of minimally polarized functional units in accordance with an embodiment ofthe invention. After obtaining a specimen, it is removed from its associated transport container followed by:
i. Placing the specimen into a stérile 50 ml conical tube containing puise rescue media and placed on rocker for 5 minutes, repeat with fresh media and container for total of three times;
ii. Removing and placing the specimen into a stérile culture dish containing puise media and excise fat and hypodermal éléments from the dermal and epidermal compartments carefully. Follicular units are left in place and are not overly dissected;
iii. Placing excised hypodermal fat components into separate a 50ml conical tube containing PRM and place in +4°C on slow rocker.
iv. Sectioning the remaining cutaneous éléments containing epidermal, follicular and dermal compartments into minimal polarized functional units (MPFUs) using ultrafine WECPREP® Blades or some form of micro-16 lancet; and
v. Placing the MPFUs components into separate a 50ml conical tube containing puise media and place in +4°C.
The following relates to secondary processing where the primary cultures are established and functional tissue éléments are prepared utilizing enzymatic préparation using conventional CLIA equipment and reagents meeting FDA and/or GM P certification:
Example 2
Example 2 is directed to processing of hypodermis and subdermal fat cellular components. Example 2 recites the following steps:
i. Spraying 70% éthanol (EtOH) on the outer side of the tissue container and placing the tissue container into laminar air flow cabinet;
ii. Sending a sample ofthe tissue or transfer medium for microbiological testing;
iii. Placing the previously washed adipose and hypodermal tissue in 150mm stérile pétri dish;
iv. Washing the tissue two times with PRM;
v. Trimming the tissue into small (3mm) pièces with stérile surgical instrument and place into stérile culture holding dish containing puise media while the dissection is completed;
vi. Aspirating media from holding dish and removing the specimen with stérile scoop or forceps followed by placing the specimen into 50 ml conical tube containing MSC Enzymatic Digestive Media, a pre-mixed digestive enzyme solution (collagénase and dispase-based), which is placed into a 37°C water bath or dry heat slow shaker and shaken for 30 minutes or until there are few particulate materials remaining;
vii. Adding 37°C phosphate buffer saline (PBS) ethylenediamine tetraacetic acid (EDTA) (equal volume PBS-EDTA) to stop the digestion;
viii. Centrifuging the suspension for 10 minutes to generate a “soft pellet;
ix. Discarding upper liquid portion and using a stérile pipette, separating the adipose population from stromal vascular fraction (SVF) in the saved mass;
x. Re-suspending the SVF in phosphate buffer saline/EDTA, PBS-EDTA (1 mM of EDTA), and re-suspending adipocyte population in PRM in two separate conical tubes;
xi. Using 100 pm stérile, filter the suspension into new stérile conical tubes;
xii. Washing the filter with PBS-EDTA;
xiii. Spin filtering the suspension for 10 minutes at room température followed by aspiration of the media and replacing the aspirated media with a known volume of fresh media;
xiv. Using a COUNTESS® automated cell counter (Thermo Fisher Scientific), count cell populations to détermine viability;
xv. Removing 20% of obtained cell population for cryopreservation with SYNTH-A-FREEZE® CTS™ (Cell Therapy Systems) from Thermo Fisher Scientific and subsequently cataloguing appropriately while using the remaining 80% population for construct assembly.
Example 3
Example 3 is directed to addition of hypodermis and subdermal fat components to the example of a construct according to an embodiment of the invention. The illustrative component addition example involves:
i. Placing a stérile NUNC® Skin Graft Cell Culture Dish or automated dish already containing the assembled and washed scaffold into a laminarflow hood and washing the scaffold again two times with puise media prior to adding cells;
ii. Inserting a label on each culture vessel with tracking number;
iii. Transferring around 5x105 to 1x106 mixed SVF cells per dish system and 1x105 adipocytes per dish;
iv. Adding a complété culture medium with or without autologous PRP as dictated by the particular requirements of a situation, to the loading réservoir;
v. Transferring the dishes into an incubator onto slow rocker for 1 hour followed by removal therefrom and resting fiat for 48 hours in separate sentinel incubator;
vi. Washing the culture medium after 48 hours, discarding the nonadherent cells, and renewing the complété culture medium. Image with a cell imaging device such as an EVOS® (ThermoFisher Scientific) and store with the designated tracking number.
vii. Every 72 hours replacing the culture medium;
viii. At confluence, washing the culture with Dulbecco's phosphatebuffered saline (DPBS) and replacing the culture media with fresh media.
ix. Placing the épithélial stem cell functional singularity constructs (ESC FSLJs) directly on the surface of the mesenchymal stem cell (MSC) construct, adding ESC media to cover both constructs, imaging the same and replacing the construct into the incubator.
x. Changing/replacing the construct media every 48 hours.
Example 4
Example 4 concerns enrichment of the minimally polarized, épithélial stem cell singularity units.
Following Example 1, the MPFUs is placed in puise rescue media in a 15 ml conical tube and spin/centrifuged into a soft pellet. The material is then subject to the following process of partial digestion:
i. Obtaining a previously aliquoted frozen 10ml digestion buffer (collagénase and dispase-based), which has been brought to room température prior adding to MPFUs;
ii. Adding the digestion solution to the soft pellet of MPFUs and gently mixing, by flicking, the tube to allow MPFUs to distribute throughout the solution;
iii. Placing the tube into 37°C water bath or dry incubator for 10 minutes;
iv. Removing the tube from the bath/incubator, gently fhcking tube and examining the content for string;
v. Having observed string, centrifuging the content into a soft pellet;
vi. Washing the cell pellet in 5-10 mL complété Defined Kératinocyte SFM medium (Keratinocyte-SFM (1X) from ThermoFisher Scientific) and centrifuging into soft pellet again;
vii. Re-suspending the pellet of activated functional singularity units in 5 mL of complété the Keratinocyte-SFM medium; and viii. Determining the cell density ofthe units using a COUNTESS® Automated Cell Counter (ThermoFisher Scientific).
Example 5
Example 5 involves adding the épithélial stem cell functional singularities (ESC aFSUs) obtained from Example 4 to a construct/scaffold. The procedure entails:
i. Placing an UPCELL™ Surface Skin Graft Cell Culture Dish already containing an assembled and washed scaffold, and to assure physiologie pH, washing the scaffold twice again with puise media prior to adding the cells;
ii. Labelling each culture vessel with a unique tracking number;
iii. Transferring ESC aFSUs to the construct via disposable transfer pipette using complété Defined Kératinocyte SFM medium (additional autologous PRP is optional);
iv. Adding the complété culture medium to a select loading réservoir and ensuring complété coverage of the construct;
v. Transferring dishes into the incubator onto slow rocker for 1 hour. Then remove from rocker and allow to remain fiat for 48 hours in separate sentinel incubator;
vi. At 48 hours, aspirating the culture medium and adding fresh Kératinocyte SFM culture medium. Imaging the culture with EVOS® and storing the culture with the assigned tracking number. Increasing the gingival fibroblasts (GF) population and viability protein and/or supplementing the PRP if a need is detected at this time.
vii. Replacing the culture medium every 48-72 hours;
viii. Upon achieving confluence, washing the culture with DPBS and replacing the media. Reducing the température using température based system of the ESC construct scaffolding to facilitate release from the dish;
ix. Placing the ESC directly on the surface of the MSC construct and adding combined media to cover both constructs. Imaging the construct, placing it back into the incubator and changing the construct media every 48 hours.
x. To confirm polarization maintenance, imaging the construct daily and adding an appropriate Cornification (rind forming) medium following confirmation that polarization has been maintained for 48 hours;
xi. Washing the construct twice with puise media at harvest and replacing the media with a defined transport media using CTS™ STEMPRO® MSC SFM base.
Example 6
Example 6 represents illustrative protocols for quality assurance and construct finalization involving cryopreservation which entails préparation the construct for shipment following defined good manufacturing processes (GMP) for cell therapy applications and include:
i. Obtaining an appropriate volume of SYNTH-A-FREEZE® cryopreservation medium (Thermo Fisher Scientific) and storing the medium at 2°C to 8°C until use;
ii. Preparing, harvesting and determining cell density using COUNTESS® Automated Cell Counter prior to centrifugation a desired quantity of cells where typical cell densities for cryopreservation with SYNTH-A-
FREEZE® medium are 5 * 105 to 3 x 106;
iii. Re-suspending the cell pellet in the pre-determined volume of 2°C to 8°C of SYNTH-A-FREEZE® medium;
iv. Immediately dispensing aliquots ofthe obtained suspension into cryovials according to the manufacturer's spécifications;
v. Placing the cryovials into an appropriate cryosystem, such as a MR.
FROSTY™ system available from Thermo Fisher Scientific Inc. that maintains freezer températures at -80°C;
vi. Transferring the vials to a liquid nitrogen long-term vapor-phase storage at -200°C to -125°C.
< 36 —-............—.....The described embodiments of the invention have-been provided in the -.....forgoing spécification, it should be understood by those skilled in the art that many modifications and embodiments of the invention will corne to mind to which the invention pertains, having benefit of the teaching presented in the foregoing s description and associated drawing. Therefore, it also should understood that the invention is not limited to the spécifie embodiments disclosed herein, and that many modifications and other embodiments ofthe invention are intended to be included within the scope of the invention. Moreover, although spécifie terms are employed herein, they are used only in generic and descriptive sense, and not for to the purposes of limiting the description invention.
Industrial Applicability
The invention relates to methods for making and methods for using constructs of micro-aggregate multicellular grafts containing isolated Leucine-rich repeat-containing G-protein coupled Receptor (LGR) expressing cells for the is delivery, application, transplantation, implantation, directed seeding, directed migration, directed tracking, in setting, laminating and/or injection ofthe cellular element generating, regenerating, enhancing and/or healing épithélial Systems, glands, hair, nerves, bone, muscle, fat, tendons, blood vessels, fascia, ocular tissues and peptide secreting cellular éléments for use in wound therapy applications, tissue engineering, cell therapy applications, regenerative medicine applications, medical/therapeutic applications, tissue healing applications, immune therapy applications, and tissue transplant therapy applications.
Claims (5)
- Claims:l. A composition, comprising (i) living LGR-expressing épithélial cells and (ii) supportive cellular entities, wherein said composition is minimally polarized, micro-aggregate, and multicellular.
- 2. The composition of claim l, further comprising growth factors.
- 3. The composition of claim 1, further comprising migratory or recruiting analytes.
- 4. The composition of claim 1, further comprising an LGR spécifie binding element selected from a ligand family, R-spondin, an epidermal-derived growth factor (EDGF), a platelet-derived growth factor (PDGF), a Wnt protein, a vascular endothélial growth factor (VEGF), an antimicrobial peptide, or combinations thereof.5. A composition of claim 1, further comprising a multi-dimensional support.6. The composition of claim 1, further comprising a delivery substrate.7. The composition of claim 6, wherein the delivery substrate is selected from scaffolding, matrix, particle, cells, collagen, fiber, or combinations thereof.8. The composition of claim 1, wherein the supportive cellular entities comprise mesenchymal cells, progenitor cells, extracellular matrix éléments, or combinations thereof.* 389. The composition of claim 1, wherein the living LGR-expressing épithélial cells are selected from LGR4-expressing cells, LGR5-expressing cells, LGR6-expressing cells, or combinations thereof.
- 5 10. A composition as defined in any one of claims 1 to 9 for use in therapy.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
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US62/086,526 | 2014-12-02 | ||
US14/954,335 | 2015-11-30 |
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OA19580A true OA19580A (en) | 2020-12-23 |
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