Patent Description:
The present 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 multi-cellular LGR construct on a delivery vector/substrate/support/scaffold for direct application.

Over the years, clinicians and researchers have 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, self-derived antimicrobial peptides, in that these agents typically function through membrane permeabilization, a mechanism less likely to lead to microbial resistance. With the continued risk of infections in wounds and the advancing epidemic of bacterial resistance to current antibiotic therapies, there is a genuine need for the development of a new class of topical antimicrobial agents for use in cutaneous burns and wounds.

There are essentially four phases of wound healing that have been described over the past century: (<NUM>) hemostasis, (<NUM>) inflammatory, (<NUM>) proliferative, and (<NUM>) remodeling. These sequential phases were first defined by the types of cells that had migrated 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 have 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 compartment, the epithelial stem cell develops from the primordial ectoderm, which later develops the more superficial epithelial compartment and, thus, also has a potential role in cutaneous wound healing. At this time, there is limited research on how transplantation and application of isolated LGR4, LGR5 and LGR6 expressing epithelial stem cells alter wound healing gene expression.

It is known that LGR4, LGR5 and LGR6 expressing epithelial stem cell populations are often destroyed following severe full-thickness damage to the skin, leaving tissues incapable of producing a viable and self-sustaining epithelial compartment. Despite a combination of granulatory and fibrotic efforts driven by localized inflammation and subsequent chemotaxis of a spectrum of cellular entities, without the epithelial stem cell focal niche, remaining tissues are left without the regenerative potential to form a functional epithelium, hair follicle, sweat gland, or the like.

Complex full thickness injuries to human and mammalian tissues and/or complex injuries involving multiple tissue elements (skin, muscle, fat, blood vessels, nerves and bone) are difficult in nature to heal. Such injuries and subsequent 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: <NUM>) cellular progenitor and/or stem cell populations; <NUM>) extracellular matrix/scaffolding elements and substrates; and <NUM>) a combination of interactions between and among cellular entities 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 essential multi-dimensional architecture required for cellular migration, differentiation, and tissue polarization. Without these cell-to-cell and cell-to-matrix interactions, remaining cellular entities within the wound bed, no matter their proliferative or lineage potential, are forced to provide primarily a barrier utility rather than develop a more complex, multi-tissue 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, cartilage, bone, muscle, blood vessels, nerves, lymphatics and related substitutes are largely based on a three part strategy: <NUM>) acquiring a tissue source and harvesting cell suspension from such tissue; <NUM>) applying these cells to a matrix or scaffold; and <NUM>) grafting the construct onto or into a target site of a human or animal. However, in the absence of the above-identified inter-dependent, 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, progenitor cell mass and proper scaffolding prevent such constructs to be useful in therapeutic applications such as multi-compartment tissue regeneration and/or bone and muscle reconstruction.

Consequently, in part due to the foregoing, substantial efforts and resources have been directed by both industry and academics to developing 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 beneficial, are often expensive and do not provide the patient with a true multi-compartment tissue construct. For example, cultured epithelial autograft (CEA) remains unable to restore both epithelial 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, epithelial 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 specific stem cell populations which are necessary to develop functional native tissues.

The inventor herein has already written about the relatively recent recognition of LGR5 and LGR6 as markers of both intestinal and epidermal stem cells in mammals. In <NPL>, Leucine-rich repeat-containing G-protein-coupled receptor (LGR) is a seven-pass transmembrane protein receptor with significant 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 <NUM> peptide significantly enhanced wound healing and reduced basal bacterial load compared with human beta defensin <NUM> and sulfadiazine. Human alpha defensin <NUM> was the only therapy to induce LGR stem cell migration into the wound bed. In addition, gene heat mapping showed significant mRNA up-regulation of key wound healing and Wnt pathway transcripts such as Wnt1 and Wisp1. So it was concluded that human alpha defensin <NUM> could be used for enhanced wound healing due to the observed increase of LGR stem cell migration into wound beds and associated bacterial reduction and hair production through the augmentation of key Wnt and wound healing transcripts. In short, this and other work led to the recognition of the potential for using LGR4+, LGR5+ and LGR6+ expressing epithelial stem cells in direct biomedical engineering soft tissue constructs.

<CIT> describes a method for culturing isolated epithelial stem cells expressing LGR5 and/or LGR6 by incubating them in the presence of a scaffold or extracellular matrix, a cell culture medium and additional factors such as R-spondin or epidermal growth factor, thereby forming organoids. The organoids can be used for the regeneration of damaged tissue.

The scope of this invention is defined by the claims. Embodiments in the description relating to methods of treatment are not covered by the claims. The more generic description of the invention is provided for illustrative purposes only. Embodiments not falling under the claims are for reference purposes only. The invention provides the following aspects:.

In this detailed description, references 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 necessarily refer to the same embodiment; however, neither are such embodiments mutually exclusive, unless so stated, and except as will be readily apparent to those skilled in the art. Thus, the invention can include any variety of combinations and/or integrations of the embodiments described herein.

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 "have", when used in this specification, specify the presence of stated features, steps, operations, elements, 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 <NUM>% by weight) as well as calcium carbonate (<NUM>%), and magnesium; by weight, bone is composed of <NUM>-<NUM>% inorganic and <NUM>-<NUM>% organic material; a portion of osseous tissue of definite shape and size, forming a part of the animal skeleton; in humans there are approximately <NUM> 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.

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 inherent to such process, method, article, or apparatus.

As used herein Epithelium means the cellular layer covering all 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, all the derivatives 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 <NUM>, G protein-coupled receptors (GPCRs) that play key roles in a variety of physiologic functions. Members of the leucine-rich GPCR (LGR) family, such as GPR48, have multiple N-terminal leucine-rich repeats (LRRs) and a <NUM>-transmembrane domain. LGR4 (Leucine-Rich Repeat Containing G Protein-Coupled Receptor <NUM>) is a Protein Coding gene. Diseases associated with LGR4 include bone mineral 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), associates 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 peritubular myoid cells. Likewise, LGR4 is required for the maintenance of intestinal stem cells and Paneth cell differentiation in postnatal intestinal crypts. LGR4 also acts as a regulator of bone formation and remodeling in addition to being involved 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 negative regulator of innate immunity by inhibiting TLR2/TLR4 associated pattern recognition and pro-inflammatory cytokine production. LGR plays an important role 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 <NUM>; BNMD17; Leucine-Rich Repeat-Containing G Protein-Coupled Receptor <NUM>; Leucine-Rich Repeat-Containing G-Protein Coupled Receptor <NUM>; and G-Protein Coupled Receptor <NUM>. External Database Identifiers for LGR4 include: HGNC: <NUM> Entrez Gene: <NUM> Ensembl: ENSG00000205213 OMIM: <NUM> and UniProtKB: Q9BXB.

As used herein LGR5 means Leucine-Rich Repeat Containing G Protein-Coupled Receptor <NUM>, a Protein Coding gene. 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. 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), associates 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, 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; G-Protein Coupled Receptor <NUM>; G-Protein Coupled Receptor <NUM>; GPR67; GPR49 and Leucine-Rich Repeat-Containing G-Protein Coupled Receptor <NUM>. External Database Identifiers for LGR5 include HGNC: <NUM> Entrez Gene: <NUM> Ensembl: ENSG00000139292 OMIM: <NUM> and UniProtKB: O75473.

As used herein LGR6 means Leucine-Rich Repeat Containing G Protein-Coupled Receptor <NUM> which is a Protein Coding gene a gene that encodes a member of the leucine-rich repeat-containing subgroup of the G protein-coupled <NUM>-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 horseshoeshaped 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 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), associates 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, 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 GPCR. External Database Identifiers for LGR6 include HGNC: <NUM> Entrez Gene: <NUM> Ensembl: ENSG00000133067 OMIM: <NUM> and UniProtKB: Q9HBX8.

As used herein Mesenchyme means an aggregation of mesenchymal cells. Primordial embryonic connective tissue consisting of mesenchymal cells, usually stellate in form, supported in inter-laminar jelly.

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 latter 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 origin (q. ), the more distal or more movable attachment is the insertion (q. ); 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 nerve cells or their processes, or that on further development will evolve into nerve cells. Referring to the dorsal side of the vertebral 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.

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 suitable anchor for 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 Pulse Rescue Media (PRM) is a formulation of a cell sustaining media mixture including Keratinocyte-SFM (1X), an antibiotic-antimycotic selected from the group consisting of penicillin, streptomycin, and amphotericin B, and fibrinogen where the Keratinocyte-SFM is composed of a mixture of epithelial cells and keratinocytes. 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.

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 equivalents may be made without departing from the scope of the invention.

<FIG> Example of flow cytometry of cell populations that exist around a hair follicle and scaffolds that such cells readily adhere to when seeded. More specifically, <FIG> 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). <FIG> is a fluorescent activated cell sorting graph with gate analysis indicating exemplary cellular markers. <FIG> depicts an array of cells types can be used to seed a spectrum of acellular matrices/substrates/scaffolds/materials.

<FIG> is a photographic representation of an example of a gross construct without micro-aggregate multi-cellular functional units containing LGR expressing stem cell foci. <FIG> depicts the construct following seeding of substrate with micro-aggregate multi-cellular functional units containing LGR expressing stem cell foci.

<FIG>, in columnar format, is an image series by differential interference contrast (DIC) confocal microscopy of LGR seeded substrates from different sources. <FIG> is a corresponding column by immunofluorescent confocal microscopy at 20x magnification of LGR6+ ESC seeded matrices of respective constructs containing LGR expressing cells. The inset white boxes represent focal zoom regions indicated in the column <FIG> while <FIG> is a column depicting the Digital merge of the respective image of <FIG> (DIC) and the immunofluorescent of <FIG> indicating matrix contour and boundaries. The columns of <FIG> respectively represent the bioluminescence measured in radiant efficiency of an acellular matrix control and a corresponding LGR6+ ESC seeded matrix at <NUM> hours post-seeding.

<FIG> depict examples of said LGR containing construct placed into living mammalian system. Placement of an LGR6+ GFP ESC Seeded Matrix Augments Healing Hair Follicle Growth. <FIG> is a 3x3 matrix of photomicrographs of <NUM> full human de-cellularized dermis thickness burn wound beds at days <NUM>, <NUM> and <NUM> containing no matrix (burn control), matrix (matrix control) and LGR6+ GFP ESC. <FIG> graphically depicts the relative expression of Cytokeratin-<NUM> transcript expression at day <NUM> of the wound beds depicted in <FIG>. 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.

<FIG> is a photomicrograph of in vivo bioluminescent imaging in murine full thickness burn wound beds at day <NUM>. <FIG> are micrographs of human dermis at 100x of the controls and LGR6+ GFP containing dermis at <NUM> hours and <NUM> hours and after seeding with ESCs. The white arrow indicates the presence of a dermal pore <FIG> provides images of the controls and the construct 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 nascent hair patches from the full thickness Nu/Nu murine wound bed.

<FIG> depict an example of said construct with LGR and supportive cellular entities following initial form of polarization as well as 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. <FIG> is confocal 20x imaging of a 5x10<NUM>RFP expressing stromal vascular fraction cellular isolate population <NUM> hours after being seeded on to a representative Adrenomedullin (ADM) (such as that available from Integra LifeSciences Corporation under the name Integra®). <FIG> is a confocal 20x image of a 5x10<NUM>FP expressing LGR6+ cellular isolate population <NUM> hours after being seeded on to a representative ADM (Integra®). <FIG> depicts confocal 20x imaging of a dual seeded representative ADM (Integra®) with 5x10<NUM>RFP expressing SVF and 5x10<NUM>GFP expressing LGR6+isolate populations <NUM> hours after being co-seeded in culture. <FIG> is of a co-seeded matrix containing 5x10<NUM>RFP expressing SVFRFP and 5x10<NUM>GFP expressing LGR6+ following <NUM> days of growth in culture. The dotted parallel lines indicate epithelial 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 correlation with LGR6+GFP and SVFRFP abundance. The arrowed "U" shaped solid line indicates a region containing a pre-seeded pore induced by a <NUM> gauge sterile needle. <FIG> is a graphical representation of the proliferation kinetics of a collagen substrate co-seeded with green LGR expressing cells and red SVF expressing cells.

<FIG> and <FIG> depict an example of a construct containing LGR cells with and without supportive cellular entities and the relative production of growth factors. Correlative Expression Profiles of Pro-angiogenic Transcripts and Protein Analytes from LGR6+GFP ESC and SVFRFP Enriched Scaffolding Culture Constructs. <FIG> graphs relative fold transcript expression (ΔΔCT) 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. Average FGF-<NUM> gene expression for co-cultured matrices was 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 intra-comparison of substrates, while the cellular entity remains constant. VEGF-A gene expression for Integra®+ LGR6+GFP ESC only vs. Dermamatrix®+ LGR6+GFP ESC only was nonsignificant (NS). <FIG> graphically represents the relative densitometric unit (RDU) of indicated protein analyte from total protein isolates: LGR6+GFP ESC (black bar), SVFRFP (grey bar), and co-cultured LGR6+GFP ESC + SVFRFP (white box) on respective scaffold substrate. (*) indicates (p-value < <NUM>), assays completed in triplicates, GAPDH housekeeping control.

<FIG> illustrate a wound/injury/void receiving therapy example of enhanced LGR cell migration, proliferation and viability into a wound namely a third degree wound bed induction and verification of the elimination of the LGR stem cell follicular bulge and adnexal structures. <FIG> depicts a wound bed template marks of <NUM> diameter. <FIG> depicts the wound bed structure at day <NUM> (the white scale bar being lmm). <FIG> illustrates an example of a 2x3 <NUM> wound bed grid. <FIG> shows topical application of the resuspended peptide at the wound site. <FIG> is a photomicrograph of H&E stain of non-burned, intact Integument/skin with hair follicle and adnexal structures. The arrow indicates the location of the magnified follicle (inset image) where the white scale bar is <NUM>. <FIG> is an H&E stain of dorsal murine skin following high temperature cautery depicting removal of epidermal, dermal and hypodermal tissues including the follicular bulge. <FIG> is DAPI/DNA stain (<NUM>',<NUM>-diamidino-<NUM>-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). <FIG> DAPI/DNA stain of dorsal murine skin following high temperature cautery depicting removal of epidermal, dermal, and hypodermal tissues including the follicular bulge where the white scale bar is <NUM>.

<FIG> depict a wound/injury/void with LGR as it relates to antimicrobial behavior over five and ten day time periods. Using <NUM> rRNA fluorescent oligonucleotide probes, in-situ hybridization indicates the presence of bacterial adhesion at the third degree burn wound bed. <FIG> presents DNA/DAPI labeling of a 3rd degree burn wound bed at day five post burn induction treated daily with SDZ. In <FIG> <NUM>'- Cy3-EUB338 labeled <NUM> rRNA of 3rd degree burn wound bed bacterial organisms (yellow grains) at day five post burn induction treated daily with SDZ are depicted. <FIG> is a digitally merged image of <FIG> corresponds to <FIG> except at day ten with DNA/DAPI labeling of 3rd degree burn wound bed treated daily with SDZ. Correspondingly, <FIG> is a photomicrograph of the <NUM>'- Cy3-EUB338 labeled <NUM> rRNA of 3rd degree burn wound bed bacterial organisms (yellow grains) at day ten post burn induction treated daily with SDZ. <FIG> is a merged image of <FIG> are images corresponding respectively to the five and ten post burn periods of <FIG> but subject to daily treatment using Defensin, alpha <NUM> (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. <FIG> 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 <NUM> rRNA labeling per unit area. <FIG> 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 <NUM> rRNA labeling per unit area. The inset graph depicts averaged white pixel intensity of <NUM> rRNA expressed in both SDZ and DEFA5 treated burn wound beds at day five using grayscale imaging software. Finally, <FIG> is a graph to illustrate averaged red channel fluorescence of <NUM> rRNA expressed in both SDZ and DEFA5 treated burn wound beds at day five. The white arrow in <FIG> indicates potential film in DEFA5 treated wound beds and the black arrow in <FIG> indicates white pixel intensity. Scale bar <NUM>. (*) indicates p-value <<NUM>.

<FIG> and <FIG> are a series of time progression photographs that represents an example of LGR expressing cellular entities within wound as it relates to augmented healing, tissue and appendage regeneration and subsequent hair growth, wound healing kinetics and nascent hair growth in treated burn wounds devoid of adnexal structures. The photographic series comprising <FIG> are gross imaging using a Leica Wild M680 surgical microscope to image healing of 3rd degree burn wound beds over <NUM> days while being treated with indicated agents MQH2O, DEFA5, DEFB1, SDZ. The white scale bar represents <NUM>. The second photographic series of <FIG> again comprises gross imaging using a Leica Wild M680 to track nascent hair growth of 3rd degree burn wound beds over <NUM> 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 <NUM>.

<FIG> 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 Graphs comprising <FIG> 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 microscopic 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 <NUM> wound beds was found to be <NUM> ± <NUM> and <NUM> ± <NUM>, respectively, compared with undetectable levels of LGR5 and LGR6 in sulfadiazine-treated wounds at day <NUM> (<FIG>, right). The magnitudes of these fold-level comparisons within human alpha defensin <NUM>-treated tissues and those specimens treated with sulfadiazine suggest that it is the absolute presence or void of cells expressing LGR5 and LGR6 migrating into the wound that defines the fold values.

Turning to the specific figures, <FIG> presents photographs of a wound area with a white scale bar representing <NUM> and the wound area calculation in black. <FIG> graphically displays the averaged wound healing rate expressed as percent % of wound area remaining over <NUM> day period of indicated topical focal agent application. The asterisk (*) represents a p-value <<NUM>. <FIG> are LGR5 and LGR6 immunofluorescent antibody labeling of a DEFA5 treated wound bed at day <NUM> where <FIG> is DNA/DAPI/Blue, <FIG> is LGR5/FITC/Green <FIG> is LGR6/TRITC/Red and <FIG> is a merger of 10C-10E. <FIG> are corresponding LGR5 and LGR6 immunofluorescent antibody labeling of SDZ (sulfadiazine) treated wound bed at day <NUM> (DNA/DAPI/Blue, LGR5/FITC/Green and LGR6/TRITC/Red). <FIG> is a merged image of <NUM>-<NUM> and includes an inset representing averaged LGR5 and LGR6 expression using Green and Red fluorescent intensity per wound bed at day <NUM>. The 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 <NUM> and again, the asterisk (*) represents a p-value <<NUM>.

<FIG> and <FIG> illustrate a wound/injury/tissue void with the LGR expressing cellular entities placed within wound as it relates to augmentation of pro-healing 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 role of human alpha defensin <NUM> versus sulfadiazine in augmenting key transcript expression within the wound. The results show that several gene subsets are significantly up-regulated within the wound beds receiving human alpha defensin <NUM> when compared with sulfadiazine therapy and that certain Wnt pathway gene subsets are significantly up-regulated in response of the LGR stem cell system to Wnt ligands in both the gut and skin.

<FIG> presents an Averaged Wound Healing RT2-PCR Array pathway heat map and corresponding gene map with fold regulation for wound beds comparing DEFA5 to SDZ treated systems. <FIG> presents an Averaged Wnt RT2-PCR Array healing pathway heat map and corresponding gene map with fold regulation for wound beds comparing DEFA5 to SDZ treated systems. The colors of the heat maps are indicated as red, more expressed in DEFA5 treated burns to green more expressed in SDZ treated burns.

<FIG> represent an example of a micro-aggregate multicellular 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/<NUM>(UBC-GFP) murine cells were isolated for cell culture expansion. <FIG> depicts LGR6 fluorescent antibody (green) expression of cells on the hair follicle following partial epidermal <NUM> unit/µL dispase digestion. (Worthington Biochemical Corp. , Lakewood, N. ) digestion for <NUM> minutes at <NUM> on a slow rocker. <FIG> is of LGR6+ cells expressing additional CD34 and CD73 markers (the arrow indicates population isolated comprising approximately <NUM> to <NUM> percent of all cells). <FIG> are eFluor450 expression histograms of an in vitro wound assay respectively showing periodic intrinsic GFP expression from C57BL/<NUM>(UBC-GFP) murine cells, CD34+ PE/Cy7 expression, LGR6+ APC expression and CD73+. The dotted lines indicates the distance of separation at <NUM>, <NUM>, and <NUM> hours following disruption of the cell layer and the scale bar = <NUM>. The graph of <FIG> sets out the averaged reduction in the distance line over time expressed as a percentage of initial distance following fluorescence sorting where the asterisk (*) represents a p-value <<NUM>.

<FIG> are photomicrographs by confocal microscopy and bioluminescence of an activated functional singularity unit (aFSU) at the time initial seeding and <NUM> 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, <FIG>.

Figures l4A-E depict an example of location LGR cellular varieties as it relates to location, phenotype, interface and polarity within a cutaneous tissue. <FIG> shows by Immunofluorescence staining, localized regions of LGR6 (Green/fluorescein isothiocyanate (FITC)) and LGR5 (Red/tetramethyl rhodamine isothiocyanate (TRITC)) expression. The scale bar is for <NUM>. <FIG> shows fluorescence-activated cell sorting isolation of the LGR6+GFP epithelial stem cells from C57BL/<NUM>(UBCGFP) murine skin with the final sort gate using LGR6+, CD34 and CD73 on the left and individual histograms depicting cellular GFP expression and correlating antibody-conjugate labels: CD73/PE-<NUM>, LGR6/Cy5, CD34/eFlour450 on the right. <FIG> shows differential interference contrast image of LGR6+GFP epithelial stem cells plated following fluorescence-activated cell sorting isolation. <FIG> depicts intrinsic GFP expression of the LGR6+GFP epithelial stem cells and <FIG> is a merged image of <FIG>. The scale bar represents <NUM>.

<FIG> 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 <FIG> depict, respectively, an initial burn template; a full thickness burn on the dorsum on Nu/Nu mouse; and delivery of Hydrogel® containing <NUM><NUM> LGR6+GFP epithelial stem cells at the base of the wound bed. The scale bar for <FIG> is <NUM>. <FIG> is an immunofluorescence image of the injection pocket DNA/DAPI-BLUE at Day <NUM> <FIG> is an immunofluorescence image of anti-LGR6/TRITC antibody labeling and <FIG> the same for LGR6+GFP epithelial stem cells. <FIG> E is a merged image of <FIG> and has a scale bar of <NUM>. <FIG> show full thickness burn wound bed induction and validation of LGR6+ stem cell engraftment into subsequent soft tissue defect.

<FIG> depict an example of LGR containing stem cell focus as it relates to delivery into and around wounds via a deliverable vector and subsequent healing, regeneration of tissues and supporting structures including but not limited blood vessel angiogenesis and/or angiogenesis. Wound healing progression following LGR6+ epithelial stem cells transplantation into full thickness wounds. The progression of wound healing is depicted following the injection of Hydrogel® from BD Biosciences, San Jose, Calif. (control) in <FIG> compared with <FIG>, LGR6+GFP epithelial stem cells seeded Hydrogel® over <NUM> days. The scale bar is <NUM>. In <FIG>, showing the implant pocket after day <NUM>, the white arrow indicates presence of a remaining LGR6+GFP epithelial stem cells population located within healing wound bed. In <FIG>, the black arrow indicates the location of the burn wound base free of LGR6+GFP epithelial stem cells.

<FIG> depicts an example of LGR containing stem cell focus following delivery into and/or around wound with subsequent healing and regeneration of tissues and related appendages such as but not limited to hair follicle and related supportive structures. <FIG> is a four panel matrix of confocal images of immunofluorescent labeled tissue specimen at day <NUM> following transplantation of LGR6+ epithelial stem cells migration into the wound bed <NUM> days. The images comprising <FIG> include DNA/DAPI-BLUE; anti-LGR6/TRITC; GFP expression of LGR6+GFP ESC.

<FIG> is a differential interference contrast image merge of all channels. The Red arrow designates regions of nascent follicle development. (See also the upper inset image). The dotted line shows epithelial polarization overlying nascent hair follicles while the white arrow indicates the location of the graft injection pocket (See also the magnification thereof in the lower inset image for an image of the initial injection pocket cellular population. The inset graph of <FIG> represents comparative KRT17/ cytokeratin <NUM> gene expression within the indicated wound beds of the control and LGR6++GFP treatment.

Referring to <FIG>, the three images are of a Transplant dome used to cover hair follicle study population burn wound beds, an LGR6++GFP ESC treated wound bed at day <NUM> (solid arrow) with nascent hair follicles (clear arrow) follicle cyst formation and a control wound bed at day <NUM>. The graph comprising <FIG> quantifies the Day <NUM> wound bed resulting from RT-PCR indicating relative gene fold expression of WNT ligands. The positive numbers indicated higher expression in LGR6+GFP epithelial stem cells wound beds while the negative numbers indicate higher expression in control wound beds.

<FIG> 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 relates to augmentation of pro-healing pathways and comparative gene expression of wounds receiving LGR6+ epithelial stem cells against a control. The graphs illustrate the relative fold expression of genes for angiogenesis, wound healing and epidermal growth factor. Correlative graphical representation of data comparing wound beds receiving LGR6+ epithelial stem cells and control therapy. As to the inset heat maps the color red indicates greater expression within the LGR6+ epithelial stem cell wound bed while the color green indicates greater expression within the control wound bed. In the bar graphs, positive numbers indicated higher expression in LGR6+GFP epithelial stem cell wound beds and negative 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 designates <<NUM> significance.

<FIG> graphically presents 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.

<FIG> illustrate an example of LGR expressing cellular foci as it relates to the regeneration of bone tissues. Isolated LGR foci can be seeded bone and remain viable. <FIG> is a gross bone image of harvested bone for culture. <FIG> is a DIC image of bone containing LGR GFP <NUM> days following seeding. <FIG> is a <NUM> Green laser confocal image of bone containing LGR6+GFP <NUM> days following seeding. It is notable that the LGR foci can undergo osteo-induction in-vitro. <FIG> depicts LGR foci following <NUM> week of osteo-induction with supplemental media. <FIG> is an Alizarin red stain of the LGR foci following osteo-induction which can undergo osteo-induction in-vitro and up regulate key osteogenic genes. Finally, <FIG> 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 <NUM> days of culture. GAPDH was used as reference standard housekeeping gene.

Use of the LGR epithelial stem cells, particularly in conjunction with a formed scaffolding substrate, provides full thickness wounds and or voids in epithelial systems with a stem cell enriched tissue substitute. Moreover, the addition of this minimally polarized functional cell unit (MPFU) to an epithelial system is enhances/improves the status of that epithelium which includes the growth, generation or regeneration 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 elements.

Recognizing that LGR4+, LGR5+ and LGR6+ stem cell and progenitor cell proliferation kinetics remain high, especially when in contact with substrate scaffolding, complete epithelial turnover rates are typically less than <NUM> days (<NUM> inter-population distance spacing). This capacity to regenerate a sufficient tissue bi-layer and subsequent barrier function suggests a role for these cells as a type of evolving biologic 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 have 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 differentiation, leading to a reduction in wound and peri-wound infections, faster wound closure, and hair follicle development.

Described herein is the translational applicability of a minimally polarized functional unit in providing an immediate, 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 epithelial tissue elements, healing and graft integration. It has been found that the minimally polarized functional unit can be applied alone, with scaffolding, soluble growth factors and/or additional cell lineage which promote the polarization of the scaffold bound populations as well s intrinsic tissue architecture required in epithelial healing and cellular regenerative efforts.

Broadly defined, the disclosed protocol involves: a) harvesting living human/mammalian tissue; b) processing the tissue element to generate 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 consisting of scaffolding, matrix, particle, cell(s) and fiber to create 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, urogenital tissues, renal tissues, reproductive tissues, lymphatic tissues, immune system tissues/elements and such related appendages and protein cellular elements.

Also disclosed is the direct delivery of supported LGR expressing epithelial stem cells by application, transplantation, implantation, directed seeding, directed migration, directed tracking, in setting, laminating and/or injection of the cellular element to alter mammalian tissue(s) in therapeutics, devices, biologics, drugs and bio-engineering.

The following is a series of examples providing an illustrative protocol sequence for practice of an embodiment of the invention.

Prior to generation 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:.

It is also recommended that an additional material referred to as Pulse Rescue Media (PRM) be produced and be available prior to commencement of the LGR aggregate extraction procedures.

Here, the PRM is direct to humans, is a cell sustaining, serum-free, media mixture Keratinocyte-SFM containing L-glutamine supplied with separately packaged prequalified human recombinant Epidermal Growth Factor <NUM>-<NUM> (EGF <NUM>-<NUM>) and Bovine Pituitary Extract (BPE) sold as Keratinocyte-SFM (1X) from Thermo Fisher Scientific to which the antibioticantimycotic agents penicillin, streptomycin, and amphotericin B are added along with a GMP- fibrinogen: human. The agent used here is Gibco® Antibiotic-Antimycotic from Thermo Fisher Scientific, a solution containing <NUM>,<NUM> units/mL of penicillin, <NUM>,<NUM>µg/mL of streptomycin, and <NUM>µg/mL of Fungizone® Antimycotic. Because the PRM is used to transport human tissues, the supplemental 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 generation and preservation of LGR expressing epithelial containing stem cell micro-aggregate functional units in accordance with an embodiment of the invention.

Example <NUM> concerns a method for extraction of minimally polarized functional units in accordance with an embodiment of the invention. After obtaining a specimen, it is removed from its associated transport container followed by:.

The following relates to secondary processing where the primary cultures are established and functional tissue elements are prepared utilizing enzymatic preparation using conventional CLIA equipment and reagents meeting FDA and/or GMP certification:.

Example <NUM> is directed to processing of hypodermis and subdermal fat cellular components. Example <NUM> recites the following steps:.

Example <NUM> is directed to addition of hypodermis and subdermal fat components to the example of a construct. The illustrative component addition example involves:.

Example <NUM> concerns enrichment of the minimally polarized, epithelial stem cell singularity units. Following Example <NUM>, the MPFUs is placed in pulse rescue media in a <NUM> conical tube and spin/centrifuged into a soft pellet. The material is then subject to the following process of partial digestion:.

Example <NUM> involves adding the epithelial stem cell functional singularities (ESC aFSUs) obtained from Example <NUM> to a construct/scaffold. The procedure entails:.

Example <NUM> represents illustrative protocols for quality assurance and construct finalization involving cryopreservation which entails preparation the construct for shipment following defined good manufacturing processes (GMP) for cell therapy applications and include:.

The described embodiments of the invention have been provided in the forgoing specification. It should be understood by those skilled in the art that many modifications and embodiments of the invention will come to mind to which the invention pertains, having benefit of the teaching presented in the foregoing description and associated drawing. Therefore, it also should understood that the invention is not limited to the specific embodiments disclosed herein, and that many modifications and other embodiments of the invention are intended to be included within the scope of the invention. Moreover, although specific terms are employed herein, they are used only in generic and descriptive sense, and not for the purposes of limiting the description invention.

Claim 1:
A composition comprising:
i) a delivery vehicle substrate selected from the group consisting of scaffolding, matrix, particle and fiber; and
ii) a micro-aggregate multi-cellular functional unit obtained by:
a) processing an isolated living mammalian skin tissue by separating fat and hypodermal elements from the isolated living mammalian skin tissue to prepare an excised mammalian tissue; and
b) sectioning the excised mammalian tissue to generate a micro-aggregate multi-cellular functional unit capable of regenerating skin tissue that includes a sectioned epidermal compartment, a sectioned dermal compartment, and a sectioned follicular compartment comprising living LGR-expressing cells.