WO2007058671A1

WO2007058671A1 - Novel uses of cells with prenatal patterns of gene expression

Info

Publication number: WO2007058671A1
Application number: PCT/US2006/013519
Authority: WO
Inventors: Michael D. West; Karen Chapman
Original assignee: West Michael D; Karen Chapman
Priority date: 2005-11-21
Filing date: 2006-04-11
Publication date: 2007-05-24

Abstract

This invention generally relates to methods to obtain mammalian cells and tissues with patterns of gene expression similar to that of a developing mammalian embryo or fetus, and the use of such cells and tissues in the treatment of human disease and age-related conditions. More particularly, the invention relates to methods for identifying, expanding in culture, and formulating mammalian pluripotent stem cells and differentiated cells that differ from cells in the adult human in their pattern of gene expression, and therefore offer unique characteristics that provide novel therapeutic strategies in the treatment of degenerative disease.

Description

NOVEL USES OF CELLS WITH PRENATAL PATTERNS OF

GENE EXPRESSION

Cross Reference to Related Applications

[0001] This application claims the benefit of United States Provisional Application Nos.: 60/670,081, filed on April 11, 2005 and 60/670,044, filed on April 11, 2005. The disclosures of these applications are hereby incorporated by reference in their entirety.

Field of the Invention

[0002] This invention generally relates to methods to obtain mammalian cells and tissues with patterns of gene expression similar to that of a developing mammalian embryo or fetus, and the use of such cells and tissues in the treatment of human disease and age-related conditions. More particularly, the invention relates to methods for identifying, expanding in culture, and formulating mammalian pluripotent stem cells and differentiated cells that differ from cells in the adult human in their pattern of gene expression, and therefore offer unique characteristics that provide novel therapeutic strategies in the treatment of burns, dermatological conditions, degenerative diseases, wound healing, and wound repair. Background of the Invention

[0003] Advances in stem cell technology, such as the use of human embryonic stem (hES), cells have become an important new subject of medical research. hES cells have a demonstrated potential to differentiate into any and all of the cell types in the human body including complex tissues. This has led to the suggestion that diseases due to the dysfunction of cells may be amenable to treatment using hES- derived cells of various differentiated types.

[0004] Wound healing may be considered unsatisfactory when there is a delay in healing, or when there is a chronic state of nonhealing, when there is an elevated rate of wound contraction, or when there is scar tissue formation. There are many situations and conditions in which scar formation is a serious unsolved complication, including surgery and recovery from deep burns. While many adult mammals close dermal wounds and deep burns with the result of permanent scar, many fetal mammals display the capacity to repair a wound far more rapidly and with little to no scarring (Burrington, 1971; Rowlatt, 1979). Longaker showed that an artificial cleft lip defect in fetal sheep could be repaired without scar formation when the surgery was done in utero (Longaker, et al, 1992). Early clinical experience with human fetal surgery revealed a remarkable observation, that the mid-gestation fetus heals surgical wounds scarlessly. Understanding the molecular pathways of scarless fetal wound healing has been the subject of considerable research. While animal models and molecular studies have uncovered some useful information regarding the differences in gene expression in wound repair in fetal vs. adult skin, researchers have not found a means of translating these observations into practical methods of scarless wound repair for postnatal human patients. Inventions which utilize gene therapy constructs to alter the pattern of gene expression in the wound to more closely mimic that of the early prenatal state, and protocols that apply growth factors to the wound in an attempt to accomplish the same goal have not resulted in a protocol with satisfactory results. [0005] The hypotheses regarding the differences in fetal vs. adult wound healing that researchers have discovered thus far include the involvement of myofibroblasts in adult wound healing. Myofibroblasts are specialized cells involved in the adult response to skin injury and they have been shown to mediate wound contraction, collagen synthesis and scarring. In an animal model of fetal wound repair, it has been shown that myofibroblasts are relatively absent in scarless wounds but are abundant in wounds that scar. The presence of myofibroblasts in skin wounds therefore appears to be associated with the development of scar. This research has also produced evidence of differences between fetal and adult connective tissue. One constituent of skin that differs in content between fetal and adult wounds is hyaluronic acid (HA), a substance critical for normal development and tissue regeneration. Scarless fetal wounds have been shown to have a relative abundance of HA as compared to adult wounds. One mechanism contributing to the normal tissue turnover of HA that has been studied is its cellular binding, internalization and subsequent degradation. These processes are mediated by specific cell-surface receptors. While receptors specific for HA binding are highly present in scar-forming adult repair processes, it has been shown that cells within scarless wounds contain significantly fewer of these HA receptors, which may contribute to the maintenance of higher concentrations of tissue HA and therefore to scarless repair. Another difference is that fetal dermal fibroblasts have been shown to express higher levels of type III and IV collagen compared to adult-derived cells (Roh et al, 2001).

Brief Description of the Figures

[0006] Figure 1 is a photograph of a representative clonogenic colony of candidate cells expressing a prenatal pattern of dermal fibroblast gene expression derived from embryoid bodies.

[0007] Figure 2 is a photograph of a representative clonogenic colony of candidate epidermal keratinocyte cells expressing a prenatal pattern of gene expression derived from embryoid bodies.

[0008] Figure 3 depicts the relative pattern of gene expression of clone 8 as compared to the standard housekeeping ADPRT gene. The following genes were expressed in clone 8, consistent with clone 8 being a dermal fibroblast progenitor cell: (a) dermo-1 (TWIST2), (b) dermatopontin (DPT), (c) PRRX2, (d) PEDF (SERPINFl), (e) AKRlCl, (f) collagen VI/alpha 3 (COL6A3), (g) microfibril- associated glycoprotein 2 (MAGP2), (h) fibulin-1 (FBLNl), (i) LOXL4, Q) CD44 (the receptor for hyaluronic acid which promotes scarless wound repair), (k) WISP2, (1) CHI3L1, (m) Odd-Skipped Related 2 (OSR2), (n) angiopoietin-like 2 (ANGPTL2), (o) RGMA, (p) EPHA5, (q) smooth muscle Actin Gamma 2 (ACTG2). The expression of the housekeeping ADPRT gene is depicted in (r). [0009] Figure 4 depicts a phase contrast photograph of dermal progenitor cells from clone 8 (ACTC51/B2). See Example 6. Series 1 also refers to Example 6.

Summary of the Invention

[0010] The present invention provides methods for the derivation, formulation, and use of cells and engineered tissues made of such cells that display a prenatal pattern of gene expression.

[0011] While techniques to differentiate hES cells into numerous differentiated states have been described, the present invention describes the novel use of hES and hED-derived cells in a relatively undifferentiated state, as opposed to a differentiated state corresponding to a postnatal state in vivo, in which the cells express a pattern of gene expression corresponding to that of the prenatal state in vivo.

[0012] In the field of the cultivation of human cells for human cell therapy, written descriptions of how these cells would be used clinically universally include a protocol for the expansion of hES or other hED cells and then the subsequent differentiation of the cells into fully differentiated cells that are subsequently injected for therapeutic effect. The present invention differs from previous methods in that the cells used therapeutically are specifically cultured and isolated under conditions that yield purified populations of cells that display a prenatal as opposed to a postnatal pattern of gene expression.

[0013] In addition, because the cells described in the present invention differ in their differentiated state from those described in previously-published methods, their unique properties can be used in novel ways for the treatment or prevention of disease.

[0014] For instance, mammalian fetal skin has been reported to differ from the skin of postnatal mammals in that prenatal skin does not scar after trauma and subsequent wound healing. The present invention provides a means of generating dermatological cells with prenatal patterns of gene expression that can be implanted into a skin wound of a postnatal mammal in order to decrease the amount of scarring and to accelerate the healing of said wound. [0015] In one aspect of the invention, the method comprises the derivation of keratinocytes with prenatal patterns of gene expression from a zygote-derived cell of a mammalian embryo produced by the fusion of a sperm and egg cell. In another aspect the present invention includes a method for the derivation of keratinocytes with prenatal patterns of gene expression from blastomere-derived cell of a mammalian embryo produced by the fusion of a sperm and egg cell, hi another aspect the invention provides for a method of derivation of keratinocytes with prenatal patterns of gene expression from a morula-derived cell of a mammalian embryo produced by the fusion of a sperm and egg cell. Another aspect includes a method of derivation of keratinocytes with prenatal patterns of gene expression from a blastocyst-derived cell of a mammalian embryo produced by the fusion of a perm and egg cell.

[0016] In one aspect of the invention, the method comprises the derivation of keratinocytes with prenatal patterns of gene expression from a zygote-derived cell of a mammalian embryo produced by nuclear transfer. In another aspect the present invention includes a method for the derivation of keratinocytes with prenatal patterns of gene expression from blastomere-derived cell of a mammalian embryo produced by nuclear transfer. In another aspect the invention provides for a method of derivation of keratinocytes with prenatal patterns of gene expression from a morula-derived cell of a mammalian embryo produced by nuclear transfer. [0017] Another aspect includes a method of derivation of keratinocytes with prenatal patterns of gene expression from a blastocyst-derived cell of a mammalian embryo produced by nuclear transfer.

[0018] In one aspect of the invention, the method comprises the derivation of keratinocytes with prenatal patterns of gene expression from a zygote-derived cell of a mammalian embryo produced by parthenogenesis. In another aspect the present invention includes a method for the derivation of keratinocytes with prenatal patterns of gene expression from blastomere-derived cell of a mammalian embryo produced by parthenogenesis. In another aspect the invention provides for a method of derivation of keratinocytes with prenatal patterns of gene expression from a morula-derived cell of a mammalian embryo produced by parthenogenesis. [0019] Another aspect includes a method of derivation of keratinocytes with prenatal patterns of gene expression from a blastocyst-derived cell of a mammalian embryo produced by parthenogenesis.

[0020] In one aspect of the invention, the method comprises the derivation of keratinocytes with prenatal patterns of gene expression from a cell line produced by the reprogramming of chromatin and subsequent incorporation of the reprogrammed chromatin into a plasma membrane. In another aspect the method comprises the derivation of keratinocytes with prenatal patterns of gene expression from a cell line produced from a blastomere. The invention also includes a method of derivation of keratinocytes with prenatal patterns of gene expression from a cell line produced by the reprogramming of chromatin and subsequent incorporation of the reprogrammed chromatin into a blastomere.

[0021] In one aspect of the invention, the method comprises the derivation of dermal fibroblasts with prenatal patterns of gene expression from a zygote-derived cell of a mammalian embryo produced by the fusion of a sperm and egg cell. In another aspect the present invention includes a method for the derivation of dermal fibroblasts with prenatal patterns of gene expression from blastomere-derived cell of a mammalian embryo produced by the fusion of a sperm and egg cell. In another aspect the invention provides for a method of derivation of dermal fibroblasts with prenatal patterns of gene expression from a morula-derived cell of a mammalian embryo produced by the fusion of a sperm and egg cell. Another aspect includes a method of derivation of dermal fibroblasts with prenatal patterns of gene expression from a blastocyst-derived cell of a mammalian embryo produced by the fusion of a perm and egg cell.

[0022] In one aspect of the invention, the method comprises the derivation of dermal fibroblasts with prenatal patterns of gene expression from a zygote-derived cell of a mammalian embryo produced by nuclear transfer. In another aspect the present invention includes a method for the derivation of dermal fibroblasts with prenatal patterns of gene expression from blastomere-derived cell of a mammalian embryo produced by nuclear transfer. In another aspect the invention provides for a method of derivation of dermal fibroblasts with prenatal patterns of gene expression from a morula-derived cell of a mammalian embryo produced by nuclear transfer. Another aspect includes a method of derivation of dermal fibroblasts with prenatal patterns of gene expression from a blastocyst-derived cell of a mammalian embryo produced by nuclear transfer.

[0023] In one aspect of the invention, the method comprises the derivation of dermal fibroblasts with prenatal patterns of gene expression from a zygote-derived cell of a mammalian embryo produced by parthenogenesis. In another aspect the present invention includes a method for the derivation of dermal fibroblasts with prenatal patterns of gene expression from blastomere-derived cell of a mammalian embryo produced by parthenogenesis. In another aspect the invention provides for a method of derivation of dermal fibroblasts with prenatal patterns of gene expression from a morula-derived cell of a mammalian embryo produced by parthenogenesis. Another aspect includes a method of derivation of dermal fibroblasts with prenatal patterns of gene expression from a blastocyst-derived cell of a mammalian embryo produced by parthenogenesis.

[0024] In one aspect of the invention, the method comprises the derivation of dermal fibroblasts with prenatal patterns of gene expression from a cell line produced by the reprogramming of chromatin and subsequent incorporation of the reprogrammed chromatin into a plasma membrane. In another aspect the method comprises the derivation of dermal fibroblasts with prenatal patterns of gene expression from a cell line produced from a blastomere. The invention also includes a method of derivation of dermal fibroblasts with prenatal patterns of gene expression from a cell line produced by the reprogramming of chromatin and subsequent incorporation of the reprogrammed chromatin into a blastomere. [0025] In one aspect of the invention, the method comprises the utilization of dermatological cells with a prenatal pattern of gene expression such as dermal fibroblasts with a prenatal pattern of gene expression that are used to improve wound healing in humans by accelerating the rate of healing, reducing the contraction of the wound following healing, and/or by reducing the amount of scar tissue formation. [0026] In another aspect of the invention, the method comprises the utilization of dermatological cells with a prenatal pattern of gene expression such as dermal keratinocytes with a prenatal pattern of gene expression that are used to improve wound healing in humans by accelerating the rate of healing, reducing the contraction of the wound following healing, and/or by reducing the amount of scar tissue formation.

[0027] More specifically, this invention provides a novel method of deriving cells with a prenatal pattern of gene expression from hES and hED cells. [0028] In one aspect of the invention, the method comprises: the utilization of dermatological cells with a prenatal pattern of gene expression such as dermal fibroblasts and dermal keratinocytes with a prenatal pattern of gene expression are used to improve wound healing in humans to prevent or reduce the amount of scarring.

[0029] In another aspect of the invention, the method comprises: the utilization of cells with a dermatological prenatal pattern of gene expression that is highly elastogenic. Dermal fibroblasts of mammalian fetal skin, especially corresponding to areas where the integument benefits from a high level of elasticity, such as in regions surrounding the joints, are responsible for synthesizing de novo the intricate architecture of elastic fibrils that function for many years without turnover. In the course of normal human aging, or in actinic skin damage, there can be a profound elastolysis of the skin resulting in an aged appearance including sagging and wrinkling of the skin.

[0030] In another aspect of the invention, the method comprises: the utilization of cells with a dermatological prenatal pattern of gene expression that also transiently express platelet-derived growth factor and are applied to wounds to promote wound healing.

[0031] In another aspect of the invention, dermal cells with a prenatal pattern of gene expression are injected into the skin of patients with epidermolysis bullosa to strengthen the skin and prevent blistering.

[0032] In another aspect of the invention, the method comprises: the utilization of cells with a dermatological prenatal pattern of gene expression for the treatment of epidermolysis bullosa. [0033] In another aspect of the invention, the method comprises: the utilization of lung connective tissue cells with a prenatal pattern of gene expression that is highly elastogenic.

[0034] In another aspect of the invention, the method comprises the utilization of cells with a prenatal pattern of gene expression that are capable of streaming throughout the brain and thereby deliver proteins for the treatment of disease.

[0035] In another aspect of the invention, the proteins expressed by the streaming cells with a prenatal pattern of gene expression are enzymes useful in the treatment of lysosomal storage diseases.

[0036] In another aspect of the invention, nonhuman animal cells with a prenatal pattern of gene expression are utilized in veterinary medicine.

Detailed Description of the Invention

Table of Abbreviations

[0037] ED Cells - Embryo-derived cells are cells derived from a zygote, blastomeres, morula or blastocyst-staged mammalian embryo produced by the fusion of a sperm and egg cell, nuclear transfer, parthenogenesis, or the reprogramming of chromatin and subsequent incorporation of the reprogrammed chromatin into a plasma membrane of an oocyte or blastomere to produce a cell line. The resulting cell line may be either a differentiated cell line or the cells may be maintained as undifferentiated ES cells. Therefore ED cells are inclusive of ES cells and cells derived by directly differentiating cells from a mammalian preimplantation embryo.

[0038] ES Cell - Embryonic stem cells derived from a zygote, blastomeres, morula or blastocyst-staged mammalian embryo produced by the fusion of a sperm and egg cell, nuclear transfer, parthenogenesis, or the reprogramming of chromatin and subsequent incorporation of the reprogrammed chromatin into a plasma membrane to produce a cell.

[0039] hED Cells - Human embryo-derived cells are ED cells derived from a human preimplantation embryo.

[0040] hES Cells - human embryonic stem cells are ES cells derived from a human preimplantation embryo. [0041] HSE - Human skin equivalents are mixtures of cells and biological or synthetic matrices manufactured for testing purposes or for therapeutic application in promoting wound repair.

[0042] ICM - Inner cell mass of the mammalian blastocyst-stage embryo.

[0043] NT - Nuclear Transfer

[0044] PS fibroblasts - Pre-scarring fibroblasts are fibroblasts derived from the skin of early gestational skin or derived from ED cells that display a prenatal pattern of gene expression with that they promote the rapid healing of dermal wounds without scar formation.

[0045] SPF - Specific Pathogen-Free

[0046] The term "pluripotent stem cells" refers to animal cells capable of differentiating into more than one differentiated cell type. Such cells include hES cells, hEDCs, and adult-derived cells including mesenchymal stem cells, neuronal stem cells, and bone marrow-derived stem cells. Pluripotent stem cells may be genetically modified or not genetically modified. Genetically modified cells may include markers such as fluorescent proteins to facilitate their identification within the egg.

[0047] The term "embryonic stem cells" (ES cells) refers to cells derived from the inner cell mass of blastocysts or morulae that have been serially passaged as cell lines. The ES cells may be derived from fertilization of an egg cell with sperm or DNA, nuclear transfer, parthenogenesis, or by means to generate hES cells with homozygosity in the MHC region.

[0048] The term "human embryonic stem cells" (hES cells) refers to cells derived from the inner cell mass of human blastocysts or morulae that have been serially passaged as cell lines. The hES cells may be derived from fertilization of an egg cell with sperm or DNA, nuclear transfer, parthenogenesis, or by means to generate hES cells with homozygosity in the HLA region.

[0049] The term "human embryo-derived cells" (hEDC) refer to morula-derived cells, blastocyst-derived cells including those of the inner cell mass, embryonic shield, or epiblast, or other totipotent or pluripotent stem cells of the early embryo, including primitive endoderm, ectoderm, and mesoderm and their derivatives, but excluding hES cells that have been passaged as cell lines. The hEDC cells may be derived from fertilization of an egg cell with sperm or DNA, nuclear transfer, parthenogenesis, or by means to generate hES cells with homozygosity in the HLA region.

[0050] The term "prenatal" generally refers to any period before full term gestational age. Cells that are prenatal according to this invention include cells that display a pattern of gene expression consistent with cells and tissues of the animal in the embryonic stage of development in vivo during the first 8 weeks of development.

[0051] The present invention provides methods for the culture of mammalian stem cells with a pattern of gene expression corresponding to that of an animal of the same species in the prenatal state in vivo, formulations of such cells useful in therapies, and the therapeutic uses of such cells and tissue formulations. [0052] The present invention also includes methods for direct differentiation of these cells from embryos without making ES cell lines (ED cells). Also, NT- derived, parthenote- derived, morula or blastomere-derived, cells that are homozygous in the HLA, those put into the gene trap system (hereby incorporate US application numbers 10/227,282 and 10/685,693), those made by dedifferentiating using cytoplasmic transfer (hereby incorporate US application numbers 10/831,599; 10/228,316; and 10/228,296). [0053] The present invention includes methods for the treatment of dermatological disease or disorders, and one such method is the derivation of dermal cells with prenatal patterns of gene expression which may be derived using any means known to those skilled in the art. Specifically this may be done by culturing embryo-derived cells, NT-derived, parthenote-derived, morula or blastomere-derived cells.

[0054] Dermal keratinocytes derived according to the invention can be grown on a biocompatible substratum and engrafted on the neodermis of artificial skin covering a wound. Autologous keratinocytes may also be cultivated on a commercially available membrane such as Laserskin™ using the methods provided in this invention. [0055] Dermal fibroblasts derived according to the invention can be grown on a biocompatible substratum and engrafted on the neodermis of artificial skin covering a wound. Autologous keratinocytes may also be cultivated on a commercially available membrane such as Laserskin™ using the methods provided in this invention.

[0056] In another embodiment of the present invention, it is possible to simplify burn treatment further and to save lives of patients having extensive burns where little or no autologous skin grafts can be repeatedly harvested in a short period of time. The dead skin tissue of a patient with extensive burns can be excised within about three to seven days after injury. The wound can be covered with any artificial skin, for example Integra™, or any dermal equivalent thereof, and dermal keratinocytes or dermal fibroblasts produced using the methods provided for in this invention may thereafter be engrafted, according to the invention, on the neodermis of the artificial skin, with resultant lower rejection and infection incidences. [0057] Epidermolysis bullosa (EB) is a group of heritable diseases that result in a loss of mechanical strength in the skin, in particular, separation of the epidermis from the dermis (blistering). EB patients have fragile skin which can blister even from mild, such as skin-to-skin, contact. These patients suffer from constant pain and scarring, which, in the worse forms, leads to eventual disfigurement, disability and often early death. EB patients lack anchors that hold the layers of their skin together and as a consequence, any activity that rubs or causes pressure produces a painful sore that has been compared to a second-degree burn. One of the forms of EB is lethal in the first weeks or months of life. Some are more long-term and cause pain and mutilation throughout the patient's lifetime. Infection is a serious, ongoing concern and no treatment for EB has been effective. To date, parents' only hope has been to attempt to protect the child's skin with gauze and ointments, to prevent and protect the wounds and healthy skin. The manifestation of the disease is highly variable depending on the locus of the mutation. Traditionally, there are three categories: the simplex form with separation within the keratinocytes, the junctional forms with separation the lamina lucida of the basement membrane, and the dystrophic forms with separation in the papillary dermis. There is now evidence of another variant at the level of hemidesmosomes and the basal cell/lamina lucida interface (Uitto, 2004).

[0058] Elastogenesis occurs in development and therefore elastogenic fibroblasts are another example of a prenatal pattern of gene expression. Examples of uses include, but are not limited to, the treatment of aging and sagging skin, vocal cords, and lung, where age-related elastolysis leads to disease or dysfunction. [0059] The cells may be combined with biological or synthetic matrices as is well known in the art. For example, dermal fibroblasts may be combined with collagen, including collagen that has been cross-linked by chemical or physical methods, and/or with other extracellular matrix components such as fibronectin, fibrin, proteoglycans, among others. The cells may be used in combination with HA.

[0060] Some embodiments of the invention provide a matrix for implantation into a patient. In some embodiments, the matrix is seeded with a population of keratinocytes or dermal fibroblast cells produced by methods according to the present invention. The matrix may contain or be pre-treated with one or more bioactive factors including, for example, drags, anti-inflammatory agents, antiapoptotic agents, and growth factors. The seeded or pre-treated matrices can be introduced into a patient's body in any way known in the art, including but not limited to implantation, injection, surgical attachment, transplantation with other tissue, injection, and the like. The matrices of the invention may be configured to the shape and/or size of a tissue or organ in vivo. The scaffolds of the invention may be flat or tubular or may comprise sections thereof. The scaffolds of the invention may be multilayered.

[0061] To form a bilayer tissue construct comprising a cell-matrix construct and a second cell layer thereon, the method additionally comprises the step of: (c) culturing cells of a second type on a surface of the formed tissue-construct to produce a bilayered or multilayered tissue construct.

[0062] An extracellular matrix-producing cell type for use in the invention may be any cell type capable of producing and secreting extracellular matrix components and organizing the extracellular matrix components to form a cell- matrix construct. More than one extracellular matrix-producing cell type may be cultured to form a cell-matrix construct. Cells of different cell types or tissue origins may be cultured together as a mixture to produce complementary components and structures similar to those found in native tissues. For example, the extracellular matrix-producing cell type may have other cell types mixed with it to produce an amount of extracellular matrix that is not normally produced by the first cell type. Alternatively, the extracellular matrix-producing cell type may also be mixed with other cell types that form specialized tissue structures in the tissue but do not substantially contribute to the overall formation of the matrix aspect of the cell-matrix construct, such as in certain skin constructs of the invention.

[0063] While any extracellular matrix-producing cell type may be used in accordance with this invention, the preferred cell types for use in this invention are derived from mesenchyme. More preferred cell types are fibroblasts, stromal cells, and other supporting connective tissue cells, most preferably human dermal fibroblasts found in human dermis for the production of a human dermal construct. Fibroblast cells, generally, produce a number of extracellular matrix proteins, primarily collagen. There are several types of collagens produced by fibroblasts, however, type I collagen is the most prevalent in vivo. Human fibroblast cell strains can be derived from a number of sources, including, but not limited to neonate male foreskin, dermis, tendon, lung, umbilical cords, cartilage, urethra, corneal stroma, oral mucosa, and intestine. The human cells may include but need not be limited to fibroblasts, but may include: smooth muscle cells, chondrocytes and other connective tissue cells of mesenchymal origin. It is preferred, but not required, that the origin of the matrix-producing cell used in the production of a tissue construct be derived from a tissue type that it is to resemble or mimic after employing the culturing methods of the invention. For instance, in the embodiment where a skin-construct is produced, the preferred matrix-producing cell is a fibroblast, preferably of dermal origin. In another preferred embodiment, fibroblasts isolated by microdissection from the dermal papilla of hair follicles can be used to produce the matrix alone or in association with other fibroblasts. In the embodiment where a corneal-construct is produced, the matrix-producing cell is derived from corneal stroma. Cell donors may vary in development and age. Cells may be derived from donor tissues of embryos, neonates, or older individuals including adults. Embryonic progenitor cells such as mesenchymal stem cells may be used in the invention and induced to differentiate to develop into the desired tissue.

[0064] Although human cells are preferred for use in the invention, the cells to be used in the method of the are not limited to cells from human sources. Cells from other mammalian species including, but not limited to, equine, canine, porcine, bovine, and ovine sources; or rodent species such as mouse or rat may be used. In addition, cells that are spontaneously, chemically or virally transfected or recombinant cells or genetically engineered cells may also be used in this invention. For those embodiments that incorporate more than one cell type, chimeric mixtures of normal cells from two or more sources; mixtures of normal and genetically modified or transfected cells; or mixtures of cells of two or more species or tissue sources may be used.

[0065] Recombinant or genetically-engineered cells may be used in the production of the cell-matrix construct to create a tissue construct that acts as a drug delivery graft for a patient needing increased levels of natural cell products or treatment with a therapeutic. The cells may produce and deliver to the patient via the graft recombinant cell products, growth factors, hormones, peptides or proteins for a continuous amount of time or as needed when biologically, chemically, or thermally signaled due to the conditions present in the patient. Either long or short- term gene product expression is desirable, depending on the use indication of the cultured tissue construct. Long term expression is desirable when the cultured tissue construct is implanted to deliver therapeutic products to a patient for an extended period of time. Conversely, short term expression is desired in instances where the cultured tissue construct is grafted to a patient having a wound where the cells of the cultured tissue construct are to promote normal or near-normal healing or to reduce scarification of the wound site. Once the wound has healed, the gene products from the cultured tissue construct are no longer needed or may no longer be desired at the site. Cells may also be genetically engineered to express proteins or different types of extracellular matrix components which are either ^λ normal" but expressed at high levels or modified in some way to make a graft device comprising extracellular matrix and living cells that is therapeutically advantageous for improved wound healing, facilitated or directed neovascularization, or minimized scar or keloid formation. These procedures are generally known in the art, and are described in Sambrook et al, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989), incorporated herein by reference. AU of the above-mentioned types of cells are included within the definition of a "matrix-producing cell" as used in this invention.

[0066] HSE using biological matrices are well known in the art and may include the use of hydrated collagen gels as described by Smola et al (1993). In brief, 4 mg/mL collagen solutions are mixed at 4 deg. C. with fibroblasts to reach a final density of 1 x 105 cells / mL. The collagen/cell suspension is then placed on a membrane such as a filter membrane and incubated for 15 min. at 37 deg C. in a humidified incubator to allow polymerization. Then the gel is placed in culture media of various compositions known in the art and allowed to contract and stabilize over time.

[0067] Synthetic polymers include polyether urethane and polyglycan, copolymers such as Polyactive a, Isotis NV, Bilthoven, the Netherlands), consisting of poly(ethyleneglycol-terephthatlate) (55%) / poly(butylene-terephthalate) (45%) (PEGT/PBT) copolymer and polyethylene glycol.

[0068] PS fibroblasts are seeded into biological or synthetic matrices at a concentration that promotes the rapid healing of wounds and/or reduces scar formation. Such concentrations range from 1.0 x 10⁵ to 1 x 10⁷ cells/cm². [0069] Other tissue such as diaphragmatic tissue may be used. [0070] In another embodiment of the invention, the cells with a prenatal pattern of gene expression are genetically modified to enhance a therapeutic effect. Such modifications may include the upregulation of expression of platelet-derived growth factor (PDGF) to improve wound repair when the modified cells are introduced into a wound. Such modifications may also include the up or down- regulation of one of a number of extracellular signaling molecules including growth factors, cytokines, extracellular matrix components, nucleic acids encoding the foregoing, steroids, and morphogens or neutralizing antibodies to such factors. Such inducers include but are not limited to: cytokines such as interleukin-alpha A, interferon-alpha AfD, interferon-beta, interferon-gamma, interferon-gamma- inducible protein- 10, interleukin-1-17, keratinocyte growth factor, leptin, leukemia inhibitory factor, macrophage colony-stimulating factor, and macrophage inflammatory protein- 1 alpha, 1-beta, 2, 3 alpha, 3 beta, and monocyte chemotactic protein 1-3, 6kine, activin A, amphiregulin, angiogenin, B-endothelial cell growth factor, beta cellulin, brain-derived neurotrophic factor, ClO, cardiotrophin-1, ciliary neurotrophic factor, cytokine-induced neutrophil chemoattractant-1, eotaxin, epidermal growth factor, epithelial neutrophil activating peptide-78, erythropioetin, estrogen receptor-alpha, estrogen receptor-beta, fibroblast growth factor (acidic and basic), heparin, FLT-3/FLK-2 ligand, glial cell line-derived neurotrophic factor, Gly-His-Lys, granulocyte colony stimulating factor, granulocytemacrophage colony stimulating factor, GRO-alpha/MGSA, GRO-beta, GRO-gamma, HCC-I, heparin-binding epidermal growth factor, hepatocyte growth factor, heregulin-alpha, insulin, insulin growth factor binding protein- 1, insulin-like growth factor binding protein- 1, insulin-like growth factor, insulin-like growth factor II, nerve growth factor, neurotophin-3,4, oncostatin M, placenta growth factor, pleiotrophin, rantes, stem cell factor, stromal cell-derived factor IB, thromopoietin, transforming growth factor- (alpha, betal, 2,3,4,5), tumor necrosis factor (alpha and beta), vascular endothelial growth factors, and bone morphogenic proteins, enzymes that alter the expression of hormones and hormone antagonists such as 17B-estradiol, adrenocorticotropic hormone, adrenomedullin, alpha- melanocyte stimulating hormone, chorionic gonadotropin, corticosteroid-binding globulin, corticosterone, dexamethasone, estriol, follicle stimulating hormone, gastrin 1, glucagons, gonadotropin, L-3,3',5'-triiodothyronine, leutinizing hormone, L-thyroxine, melatonin, MZ-4, oxytocin, parathyroid hormone, PEC-60, pituitary growth hormone, progesterone, prolactin, secretin, sex hormone binding globulin, thyroid stimulating hormone, thyrotropin releasing factor, thyroxin- binding globulin, and vasopressin, extracellular matrix components such as fibronectin, proteolytic fragments of fibronectin, laminin, tenascin, thrombospondin, and proteoglycans such as aggrecan, heparan sulphate proteoglycan, chontroitin sulphate proteoglycan, and syndecan. [0071] In another embodiment, the cells of the present invention display a prenatal pattern of gene expression consistent with dermal fibroblast progenitor cell. For example, the cells of the invention express the following prenatal gene expression profile, including, but not limited to, dermo-1 (TWIST2), dermatopontin (DPT), PRRX2, PEDF (SERPINFl), AKRlCl, collagen VI alpha 3 (COL6A3), microfibril- associated glycoprotein 2 (MAGP2), Fibulin-1 (FBLNl), LOXL4, CD44, WISP2, CHBLl₅ Odd-Skipped Related 2 (OSR2), angiopoietin-like 2 (ANGPTL2), RGMA, EPHA5, and Actin Gamma 2 (ACTG2).

Applications

[0072] It is envisioned that the disclosed methods for the culture of animal tissues are generally useful in mammalian and human cell therapy, such as human cells useful in treating derniatological and blood cell disorders.

[0073] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present specification, including definitions, will control. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Generally, nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics, developmental biology, cell biology described herein are those well-known and commonly used in the art. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention.

[0074] All publications and other references mentioned herein are incorporated by reference in their entirety. Although a number of documents are cited herein, this citation does not constitute an admission that any of these documents forms part of the common general knowledge in the art. Throughout this specification and claims, the word "comprise," or variations such as "comprises" or "comprising" will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. [0075] In order for that this invention may be better understood, the following examples are set forth. These examples are for purposes of illustration only and are not be construed as limiting the scope of the invention in any matter.

Example 1

[0076] hES cells are grown to form embryoid bodies (EB) (see, e.g., US application number 60/538,964, filed 1/23/2004, international patent publication no. WO05070011, published 8/4/2005 and U.S. patent publication no. 20060018886, published 1/26/2006, the disclosure of each of whcih is hereby incorporated by reference in its entirety) and plated out to form fibroblast-like cells that express dermal gene expression. The fibroblast-like cells are then plated at limiting dilution and expanded from a single cell by any method known to those skilled in the art, (e.g. non-limiting example includes using cloning cylinders). These single cell-derived clones can then be expanded, cryopreserved, quality controlled, and their pattern of gene expression tested using gene expression arrays as is well known in the art. For scarless skin repair, the pattern of gene expression, such as that of the TGF -beta pathway, that is present in fetal skin of the first two trimesters is known in the art and is used as a marker of cells useful in scarless skin repair. Alternatively, dermal fibroblasts can be isolated that express proteins for elastogenesis useful in inducing elastogenesis when transplanted in vivo.

Example 2

[0077] hES cell colonies from one six well plate were grown to form embryoid bodies (EB) (see, e.g., US application number 60/538,964, filed 1/23/2004, international patent publication no. WO05070011, published 8/4/2005 and U.S. patent publication no. 20060018886, published 1/26/2006, the disclosure of each of whcih is hereby incorporated by reference in its entirety) and plated out to form fibroblast-like cells that express dermal gene expression.

[0078] Specifically, colonies from the hES cell line H9 were differentiated using in situ colony differentiation by the removal of LIF-containing medium and the addition of DMEM medium containing 10% FBS. After 5 days of exposure to differentiation medium), the cells were trypsinized, and plated onto bacteriological plates and cultured for an additional 20 days to further induce differentiation as embryoid bodies. The cells were then trypsinized for 10 minutes with 0.25% tryspin/EDTA, neutralized with DMEM medium containing 10% FBS, counted with a Coulter counter, and the cells were plated at limiting dilutions from 5,000 plated cells, to 2,000 cells to 5,00 cells introduced into the 15 cm tissue culture plates with DMEM medium supplemented with 10% FBS with the plates coated with the extracellular matrix material Matrigel as per the manufacturer's instructions and subsequently the cells were incubated in 5% ambient oxygen undisturbed for two weeks.

[0079] Clonal colonies were identified by phase contrast microscopy and those that are uniformly circular and well separated from surrounding colonies were marked for removal using cloning cylinders. A representative clonogenic colony of candidate cells expressing a prenatal pattern of dermal fibroblast gene expression derived from embryoid bodies is shown in Figure 1. [0080] The trypsinized cells from within each cloning cylinder are then replated into collagen coated 24 well plates and incubated until the cells reach confluency. Those that grow at a relatively rapid rate of approximately one doubling a day are then karyotyped and determined to be normal human. A total genomic expression analysis using the Illumina system is then performed on the cells. [0081] For scarless skin repair, the pattern of gene expression, such as that of the TGF-beta pathway, that is present in fetal skin of the first two trimesters is known in the art and is used as a marker of cells useful in scarless skin repair. Alternatively, dermal fibroblasts can be isolated that express proteins for elastogenesis useful in inducing elastogenesis when transplanted in vivo.

Example 3

[0082] hES cell colonies from one six well plate were grown to form embryoid bodies (EB) (see, e.g., US application number 60/538,964, filed 1/23/2004, international patent publication no. WO05070011, published 8/4/2005 and U.S. patent publication no. 20060018886, published 1/26/2006, the disclosure of each of whcih is hereby incorporated by reference in its entirety) and plated out to form epidermal keratinocytes that express a prenatal pattern of gene expression. [0083] Specifically, colonies from the hES cell line H9 were differentiated by the removal of LIF-containing medium and the addition of DMEM medium containing 10% FBS. After 5 days of exposure to differentiation medium, the cells were trypsinized, and plated onto bacteriological plates and cultured for an additional 20 days to further induce differentiation as embryoid bodies. The cells were then trypsinized for 10 minutes with 0.25% tryspin/EDTA, neutralized with DMEM medium containing 10% FBS, counted with a Coulter counter, and the cells were plated at limiting dilutions from 5,000 plated cells, to 2,000 cells to 500 cells introduced into the 15 cm tissue culture plates with EpiLife medium (Cascade Biologies) Cat# M-EP/cf medium supplemented with calcium, LSGS (Cat#S-003- 10) and recombinant collagen (Cat#R-011-K) per manufacturer's instructions. The cells were subsequently incubated in 5% ambient oxygen undisturbed for two weeks.

[0084] Clonal colonies were identified by phase contrast microscopy and those that are uniformly circular and well separated from surrounding colonies were marked for removal using cloning cylinders. A representative colony is shown in Figure 2.

[0085] The trypsinized cells from within each cloning cylinder are then replated into collagen coated 24 well plates and incubated in the same medium until the cells reach confluency. Those that grow at a relatively rapid rate of approximately one doubling a day are then karyotyped to determine that they are normal human. A total genomic expression analysis using the Illumina system is then performed on the cells.

[0086] For improved wound repair, the keratinocytes with robust proliferative capacity are combined with dermal fibroblasts with a prenatal pattern of gene expression to produce skin equivalents capable of imparting a regenerative capacity to postnatal skin.

Example 4

[0087] Non-human ES cells are grown to form embryoid bodies (EB) (see, e.g., US application number 60/538,964, filed 1/23/2004, international patent publication no. WO05070011, published 8/4/2005 and U.S. patent publication no. 20060018886, published 1/26/2006, the disclosure of each of whcih is hereby incorporated by reference in its entirety) and plated out to form fibroblast-like cells that express dermal gene expression. The fibroblast-like cells are then plated at limiting dilution and expanded from a single cell by any method known to those skilled in the art, (e.g. non-limiting example includes using cloning cylinders). These single cell-derived clones can then be expanded, cryopreserved, quality controlled, and their pattern of gene expression tested using gene expression arrays as is well known in the art. For scarless skin repair, the pattern of gene expression, such as that of the TGF-beta pathway, that is present in fetal skin of the first two trimesters is known in the art and is used as a marker of cells useful in scarless skin repair. Alternatively, dermal fibroblasts can be isolated that express proteins for elastogenesis useful in inducing elastogenesis when transplanted in vivo.

Example 5

[0088] hES cells are grown to differentiate without forming embryoid bodies (EB) (see, e.g., US application number 60/538,964, filed 1/23/2004, international patent publication no. WO05070011, published 8/4/2005 and U.S. patent publication no. 20060018886, published 1/26/2006, the disclosure of each of whcih is hereby incorporated by reference in its entirety) and plated out to form fibroblast-like cells that express dermal gene expression. The fibroblast-like cells are then plated at limiting dilution and expanded from a single cell by any method known to those skilled in the art, for instance by using cloning cylinders. These single cell-derived clones can then be expanded, cryopreserved, quality controlled, and their pattern of gene expression tested using gene expression arrays as is well known in the art. For scarless skin repair, the pattern of gene expression, such as that of the TGF-beta pathway, that is present in fetal skin of the first two trimesters is known in the art and is used as a marker of cells useful in scarless skin repair. Alternatively, dermal fibroblasts can be isolated that express proteins for elastogenesis useful in inducing elastogenesis when transplanted in vivo.

Example 6

[0089] Colonies from the hES cell line ACT3 were differentiated using in situ colony differentiation by the removal of LIF-containing medium and the addition of DMEM medium containing 10% FBS. After various periods of time (5, 7, and 9 days of exposure to differentiation medium), the cells were trypsinized, and plated onto 15 cm plates coated with the extracellular matrix protein collagen, and cultured for an additional 20 days to further induce differentiation. The cells were then trypsinized and counted with a Coulter counter, and the cells were plated at increasing dilutions with a volume containing 2,500 cells, 5,000 cells and 25,000 cells introduced into the 15 cm tissue culture plates and subsequently incubated in 5% ambient oxygen undisturbed for two weeks.

[0090] Clonal colonies were identified by phase contrast microscopy and those that are uniformly circular and well separated from surrounding colonies were marked for removal using cloning cylinders.

[0091] The trypsinized cells from within each cloning cylinder were then replated into collagen coated 24 well plates and incubated. Of 61 colonies isolated, 29 grew at a relatively rapid rate of approximately one doubling a day. The cells were karyotyped and determined to be normal human. A total genomic expression analysis using the Illumina system was performed on the cells. [0092] Of the 17 colonies, clone 8 displayed a pattern of gene expression consistent with dermal fibroblast progenitors with its expression of dermo-1 (TWIST2), dermatopontin (DPT), PRRX2 (which is a marker of fetal scarless wound repair (J Invest Dermatol 11 l(l):57-63 1998)), PEDF (SERPINFl)₅ AKRlCl, collagen VI/alpha 3 (COL6A3), microfibril- associated glycoprotein 2 (MAGP2), which is a component of elastin-associated microfibrils, a component associated with elastogenesis Fibulin-1 (FBLNl). In developing prenatal skin, the MAGP2 protein is detected in the deep dermis and around hair follicles. The expression of MAGP2 has been reported to be up to six-fold higher in the prenatal state than postnatal and its expression precedes elastin synthesis in development (Gibson et al, J. Histochem. Cytochem. 46(8): 871-886 (1998)), GLUT5, WISP2, CHI3L1, Odd-Skipped Related 2 (OSR2), angiopoietin-like 2 (ANGPTL2), RGMA, EPHA5, the receptor for hyaluronic acid which promotes scarless wound repair (CD44), and a relative lack of the smooth muscle actins of a myofibroblast such as Actin Gamma 2 (ACTG2) (see Figure 3).

[0093] Markers that uniquely identify dermal progenitors from this region of the developing dermis include the positive expression of TWIST2, DPT, PRRX2, MAGP2, and WISP2 at levels comparable to ADPRT as shown in Figure 1, and the relative lack of expression of ACTG2 in relation to ADPRT as shown. A phase contrast photograph of the dermal fibroblast progenitors is shown in Figure 4. All levels of gene expression were compared to the internal reference expression of the housekeeping ADPRT gene.

[0094] The relatively abundant expression of EPHA5 and RGMA in these dermal progenitors promote neuronal outgrowth and innervation of the forming tissues, are therefore useful in regenerating skin while promoting the innervation of the skin graft with sensory neurons and is an example of genes not expressed at comparable levels postnatally . The relatively abundant expression of angiopoietin- Iike2 (ANGPTL2) is another example of dermal cells with a prenatal pattern of gene expression, able to promote vascularization.

Example 7

[0095] Nonhuman ES cells are grown to differentiate without forming embryoid bodies (EB) and plated out to form fibroblast-like cells that express dermal gene expression. The fibroblast-like cells are then plated at limiting dilution and expanded from a single cell as is well known in the art, for instance by using cloning cylinders. These single cell-derived clones can then be expanded, cryopreserved, quality controlled, and their pattern of gene expression tested using gene expression arrays as is well known in the art. For scarless skin repair, the pattern of gene expression, such as that of the TGF-beta pathway, that is present in fetal skin of the first two trimesters is known in the art and is used as a marker of cells useful in scarless skin repair. Alternatively, dermal fibroblasts can be isolated that express proteins for elastogenesis useful in inducing elastogenesis when transplanted in vivo.

Example 8

[0096] hES cells are injected into an animal to induce three dimensional growth, including but not limited to immunocompromized animals such as nude mice, or into SPF embryonated chick eggs to induce teratoma formation in avian species. From these teratomas, dermal fibroblasts and keratinocytes are plated out to form cells that express dermal gene expression, such as that above for scarless skin repair or elastogenesis. Example 9

[0097] Nonhuman ES cells are injected into an animal to induce three dimensional growth, including but not limited to immunocompromized animals such as nude mice, or into SPF embryonated chick eggs to induce teratoma formation in avian species. From these teratomas, dermal fibroblasts and keratinocytes are plated out to form cells that express dermal gene expression, such as that above for scarless skin repair or elastogenesis.

Claims

WE CLAIM:

1. Purified mammalian cells that display a prenatal pattern of gene expression.

2. The purified mammalian cells according to claim 1, wherein said cells are dermal fibroblast progenitor cells and wherein said cells express the following markers: dermo-1 (TWIST2), dermatopontin (DPT), PRRX2, PEDF (SERPINFl), AKRlCl, collagen VI alpha 3 (COL6A3), microfibril- associated glycoprotein 2 (MAGP2), Fibulin-1 (FBLNl), LOXL4, CD44, WISP2, CHI3L1, Odd-Skipped Related 2 (OSR2), angiopoietin-like 2 (ANGPTL2), RGMA, EPHA5, and Actin Gamma 2 (ACTG2).

3. The method of claim 1 or claim 2, wherein the mammalian cells with a prenatal pattern of gene expression are human.

4. The method of claim 1 or claim 2, wherein the mammalian cells with a prenatal pattern of gene expression are feline or canine.

5. A method for the treatment of disease in a mammalian species wherein the cells of claim 1 or claim 2 are injected into a mammal for the treatment or prevention of disease, wherein the cells are introduced into a wound to facilitate wound healing.

6. A method for the treatment of disease in a mammalian species wherein the cells of claim 1 or claim 2 are injected into a mammal for the treatment or prevention of disease, wherein the cells are introduced into a wound to facilitate scarless wound repair.

7. A method for the treatment of disease in a mammalian species wherein the cells of claim 1 or claim 2 are injected into a mammal for the treatment or prevention of disease, wherein the cells are introduced into the skin to deposit elastin.