WO2021097246A1 - Methods and products for treating renal disease - Google Patents

Abstract

The invention relates to secreted stem cell factors for tissue modulation and related methods. The secreted stem cell factor is produced and secreted by ABCB5+ stem cells. In particular methods for treating renal disease using the factor are provided.

Description

METHODS AND PRODUCTS FOR TREATING RENAL DISEASE

RELATED APPLICATIONS

This application claims the benefit of priority under 35 U.S.C. § 119(e) to U.S. Provisional Application No. 62/936,346, filed on November 15, 2020 and entitled

“METHODS AND PRODUCTS FOR TREATING RENAL DISEASE” which is herein incorporated by reference in its entirety.

BACKGROUND OF INVENTION

Renal diseases represent a global public health problem affecting a large number of patients worldwide. Due to their essential role in homeostasis maintenance, kidneys are exposed to numerous insults of different nature, resulting in a large variety of renal injuries that can affect the lifestyle of patients to varying extents. Early stage diagnosis of kidney damage is essential to be able to intervene immediately and efficiently. Diagnosis of kidney diseases is possible by checking markers of renal failure in blood and urine. Glomerular filtration rate (GFR), which describes the efficiency of renal filtration, is considered the best indicator of renal function and any changes to this parameter may reflect a pathological state. However, since early kidney diseases usually do not have any symptoms, patients often have a delayed diagnosis, resulting in a poor prognosis. Early detection and treatment of acute kidney injury (AKI) is extremely important to prevent its possible progression to chronic kidney disease or end-stage renal disease (ESRD). Regarding possible treatments, the only available options to date for ESRD are dialysis and/or renal transplantation. These are invasive and expensive.

SUMMARY OF THE INVENTION

The invention in some aspects relates to products produced by stem cells which have unique properties for treating diseases such as renal disease. In some aspects the invention is a composition, comprising a secreted stem cell factor (CSCF), wherein the CSCF is isolated from a population of ABCB5+ stem cells. In some embodiments the CSCF is present in an ABCB5+ cell derived conditioned medium. In other embodiments the ABCB5+ cell derived conditioned medium is isolated from a population of ABCB5+ stem cells co-cultured with macrophage. In some embodiments the ABCB5+ cell derived conditioned medium is isolated from a population of ABCB5+ stem cells stimulated with IFN-γ. In some embodiments the ABCB5+ cell derived conditioned medium is isolated from a population of ABCB5+ stem cells stimulated with lipopolysaccharide (LPS).

In some embodiments the ABCB5+ stem cells are ABCB5+ dermal stem cells. In other embodiments the ABCB5+ stem cells are ABCB5+ limbal stem cells. In yet other embodiments, the ABCB5+ stem cells are synthetic ABCB5+ stem cells.

The ABCB5+ stem cells may be ABCB5+ dermal mesenchymal stem cells. In some embodiments at least 85% or 90% of the population of stem cells are ABCB5+ stem cells.

In some embodiments of the invention, ABCB5(+) stem cells are limbal or retinal stem cells. ABCB5(+) stem cells may be obtained from (e.g., isolated from or derived from) the basal limbal epithelium of the eye or from the retinal pigment epithelium (RPE). In some embodiments, ABCB5(+) stem cells are obtained from human eye. Other ABCB5(+) stem cell types such as, for example, those obtained from the central cornea may be used in various aspects and embodiments of the invention.

A method for preparing a composition of a secreted stem cell factor (CSCF), is provided in other aspects of the invention. The method involves culturing a population of ABCB5+ stem cells for at least two days after the cells are added to a dish for culturing (seeding day), isolating a culture medium from the cell population, and formulating the isolated culture medium as a composition of a CSCF. In some embodiments the method further comprises purifying the CSCF from the isolated culture medium.

In some embodiments the ABCB5+ stem cells are co-cultured with macrophage. The macrophage may be a human monocyte cell line THP-1 cultured with PMA to induce macrophage differentiation and polarized to form Ml pro-inflammatory macrophages. In some embodiments the ABCB5+ stem cells are stimulated with IFN-γ. In some embodiments 50IU/ml IFN-γ is added to the ABCB5+ stem cells on the seeding day. In other embodiments the ABCB5+ stem cells are stimulated with lipopolysaccharide (LPS). In some embodiments 50IU/ml IFN-γ and 20ng/ml LPS is added to the ABCB5+ stem cells one day after the seeding day.

In some embodiments the ABCB5+ stem cells are cultured for three days following the seeding day and the culture medium is isolated on the third day. In some aspects the invention is a method for treating renal disease in a inducing liver tissue generation in a subject in need thereof, by injecting a composition comprising a secreted stem cell factor (CSCF), wherein the CSCF is isolated from a population of ABCB5+ stem cells into the subject in an effective amount to treat renal disease. In some embodiments the composition is administered systemically into the subject by intravenous or intraperitoneal delivery.

In some embodiments the conditioned medium is further processed to separate the CSCF from other components. In some embodiments the CSCF is isolated and purified from the conditioned medium. In some embodiments the CSCF is a polypeptide such as a cytokine. In some embodiments the CSCF is an exosome.

Use of a population of stem cells of the invention for treating renal disease as described herein is also provided as an aspect of the invention.

A method for manufacturing a medicament of a conditioned medium or a secreted stem cell factor (CSCF) of the invention for treating the disorders as described herein is also provided.

Each of the limitations of the invention can encompass various embodiments of the invention. It is, therefore, anticipated that each of the limitations of the invention involving any one element or combinations of elements can be included in each aspect of the invention. This invention is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings. The invention is capable of other embodiments and of being practiced or of being carried out in various ways. Also, the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of "including," "comprising," or "having," “containing”, “involving”, and variations thereof herein, is meant to encompass the items listed thereafter and equivalents thereof as well as additional items.

BRIEF DESCRIPTION OF DRAWINGS

The accompanying drawings are not intended to be drawn to scale. In the drawings, each identical or nearly identical component that is illustrated in various figures is represented by a like numeral. For purposes of clarity, not every component may be labeled in every drawing. In the drawings: Fig. 1 shows the experimental set-up for the establishment of cisplatin-induced nephropathy model in SD rats.

Fig. 2 is a schematic depicting conditioned media obtained by collecting the supernatant of ABCB5+ cells and/or macrophages with or without stimuli. IFN-γ: Interferon-gamma; LPS: lipopolysaccharide

Fig. 3 shows the experimental set-up for the assessment of the therapeutic potential of human ABCB5+ cells and conditioned media in a cisplatin-induced nephropathy model

Fig. 4 shows the effect of different treatments on a cisplatin-induced nephrotoxicity model in plasma creatinine and urea concentration. Statistically significant differences (treatment vs cisplatin) are indicated as *p< 0.05 and **p< 0.005.

Fig. 5 shows the effect of different treatments on a cisplatin-induced nephrotoxicity model in plasma GLDH level. Statistically significant differences (treatment vs cisplatin) are indicated as *p< 0.05 and **p< 0.005.

Fig. 6 shows albuminuria found by different treatments on a cisplatin-induced nephrotoxicity mode. Values were normalized to the volume of urine produced in 16 hours. Statistically significant differences (treatment vs cisplatin) are indicated as *p< 0.05 and **p< 0.005.

Fig. 7 shows glycosuria found by different treatments on a cisplatin-induced nephrotoxicity model. Values were normalized to the volume of urine produced in 16 hours. Statistically significant differences (treatment vs cisplatin) are indicated as *p< 0.05 and **p< 0.005.

Fig. 8 shows a transcutaneous assessment of renal function (ABZWCY-HPβCD half-life). Effect of different treatments on a cisplatin-induced nephrotoxicity model. Statistically significant differences (treatment vs cisplatin) are indicated as *p< 0.05 and **p< 0.005.

Fig. 9 shows a GSEA analysis using KEGG database: number of significant (adjusted p-value< 0.05) upregulated (upper Fig.) and downregulated (lower Fig.) pathways in the treatment groups.

Fig. 10 shows the results of an ELISA of 4 secreted cytokines: IL6, TNFα, G- CSF and ΤGFβ1. Data are corrected for the negative control and adjusted for the dilution factor. All samples were assayed in duplicate and values are shown are means ± SD from 2-3 independent test.

DESCRIPTION OF THE INVENTION

Stem cell therapy represents a promising approach for the treatment of several pathologies in different medical fields, including nephrology. Kidney diseases are a global public health problem with a lack of effective therapies to prevent progressive loss of renal function after initial damage. In some instances, the only available treatments are dialysis or renal transplantation. Stem cell therapy has been suggested as a promising approach for the treatment of renal injuries. A number of inconclusive studies have been conducted but no therapeutic regimen has been developed.

A well-defined animal model of cisplatin-induced kidney injury has been developed herein. This model was then used to assess the therapeutic potential of ABCB5+ cells, as well as factors secreted by the cells (also referred to herein as conditioned media). The progression of the renal damage was evaluated by periodically measuring plasmatic and urinary parameters, performing transcutaneous assessment of renal function and checking the metabolic parameters, cytokine analysis, and both mRNA and miRNA expression profiling.

The results showed that when animals were treated with stem cells, a significant amelioration in the plasma and urine parameters, renal function and histology were not observed. The cells were administered by intravenous (iv) and intraperitoneal (i). However, genomic changes in the animals in response to stem cell therapy were observed. Specifically, the ip administered ABCB5+ cells triggered new cell renal cell formation and promoted a higher reduction of inflammation compared to the iv administered ABCB5+ cells.

In contrast, and quite surprisingly it was discovered that factors secreted by ABCB5 + stem cells were sufficient (delivered in the absence of the cells) to demonstrate therapeutic potential both at a functional and a genomic level in treating nephrotoxicity. When three different conditioned media were administered to the animals, changes at the functional level were readily apparent. Indeed, markers of renal function, such as plasma creatinine and urea and urine albumin, were ameliorated especially after treatment with secreted factors isolated from untreated or IFN-g and LPS stimulated ABCB5+ stem cells (CM and CM+ respectively), while animals which received secreted factors from a mixture of ABCB5+ stem cells and macrophage (coCM+) did not show any deviation from the control animals. A similar trend was observed when the transcutaneous assessment of renal function was performed. Additionally, CM and CM+ treated animals did not show the typical loss of weight observed in the other experimental groups. The coCM+ treated animals did, however, exhibit changes at the genomic level. That group of animals exhibited better results in terms of reduction of inflammation and new cell formation than others. The animals that were treated with secreted factors isolated from unstimulated ABCB5+ stem cells (CM treated group) demonstrated both a reduction of inflammation and downregulation of miRNAs associated with metabolic processes, cell growth and response to wounding.

The results presented herein demonstrate that a composition comprising secreted factors from ABCB5+ stem cells displayed a significant therapeutic potential both at functional and genomic level in treating nephrotoxicity. The fact that such factors displayed this property when the cells which secrete the factors were less effective, was surprising.

Thus, in some aspects the invention relates to compositions comprising factors secreted from ABCB5+ stem cells. Factors secreted from stem cells are referred to herein as secreted stem cell factors (CSCFs). A CSCF is a component or mixture of components that are isolated from a population of ABCB5+ stem cells. In some embodiments the CSCF is conditioned medium which is isolated from cultured ABCB5+ stem cells, either alone or mixed with other cells. In some embodiments the ABCB5+ cell derived conditioned medium is isolated from a population of ABCB5+ stem cells stimulated with stimulatory factors such as IFN-γ and/or lipopolysaccharide (LPS). In other embodiments the ABCB5+ stem cells are cultured without the addition of stimulatory agents.

The CSCF may comprise the whole conditioned medium sample. As used herein "conditioned media" is media prepared by the culture of ABCB5+ stem cells, wherein the cells are natural or genetically-modified. The conditioned media includes proteins and other compositions representative of the secretome of the living material grown therein. The cell secretome refers not only to the collection of proteins that contain a signal peptide and are processed via the endoplasmic reticulum and Golgi apparatus through the classical secretion pathway, but encompasses proteins shed from the cell surface and intracellular proteins released through non-classical secretion pathway or exosomes. These secreted proteins may be enzymes, growth factors, cytokines, hormones, and/or other soluble mediators. The medium used to produce the conditioned media by growth of living cells or tissue therein may be any medium suitable for growth of the living cells or tissue. A large variety of media is available commercially, and one of ordinary skill in the art could determine a useful or optimal medium for use in this context. In one aspect, the media is serum-free. Various other fractionation processes, such as precipitation, centrifugation, affinity separation, or filtration may be applied to clean up, to remove harmful compounds, or to otherwise fractionate the conditioned media. One skilled in the art will be familiar with producing conditioned media, and the incubation time in order to condition the media can range from 1-5 days depending on the type of cells growing in the media and their concentration within the media. In some embodiments, the cells have been growing in the media for at least 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 hours, 60 hours, or 72 hours. Each possibility represents a separate embodiment of the invention.

Alternatively one or more components may be isolated and purified from the conditioned medium. For instance, the CSCF may be a polypeptide found in the conditioned medium. One or more polypeptides maybe separated from the conditioned medium and used as the CSCF using techniques known in the art. For instance, techniques such as removal of non-proteins (i.e. nucleic acid degradation) together with chromatography (i.e. liquid chromatography (LC), and fast protein LC and high- performance LC) are useful. Additionally specific proteins may be isolated from the medium using binding interactions with antibodies, specific nucleic acids or aptamers or alternatively based on size using combinations of mass spec and chromatography.

In some embodiments the CSCF is a polypeptide that is a cytokine. For instance, the CSCF may be selected from ΤGFβ1, TNFα, IL6 and/or G-CSF levels.

In other embodiments the CSCF is a nucleic acid, a small molecule and/or an exosome. For instance, nucleic acids (DNA or RNA), can be purified using selective adsorption techniques such as silica residues with positive charges, since nucleic acids have negative charges from their phosphodiester bonds. Specialized methods of nucleic acid purification, such as miniprep and midiprep, based on the fact that all nucleic acid molecules are highly negatively charged are also useful. Exosomes may be purified out of conditioned medium using reagents which rely on binding up water molecules, which forces less-soluble components such as vesicles out of solution, allowing them to be collected by a short, low-speed centrifugation. Such reagents are commercially available from companies such as ThermoFisher Scientific (Total Exosome Isolation Reagent).

"Exosome", as used herein, refers to cell-derived extracellular vesicles of endocytic origin, with a size of 50-100 nm, and secreted from stem cells. “Microextracellular vesicles”, as used herein, refers to cell-derived extracellular vesicles originating from the plasma membrane, with a size of 100-1000 nm, and secreted from stem cells.

The cells useful for making the conditioned medium of the invention are ABCB5 positive stem cells. ABCB5 is a novel and important marker for the isolation of multipotent stem cell populations from normal human tissue. “ABCB5(+) stem cells,” as used herein, refers to cells having the capacity to self-renew and to differentiate into mature cells of multiple adult cell lineages. These cells are characterized by the expression of ABCB5 on the cell surface. In some embodiments of the invention, ABCB5(+) stem cells are dermal or ocular stem cells. In other embodiments the ABCB5(+) stem cells are synthetic stem cells.

“ABCB5 positive dermal mesenchymal stem cells” as used herein refers to cells of the skin having the capacity to self-renew and to differentiate into mature cells of multiple adult cell lineages such as bone, fat and cartilage. These cells are characterized by the expression of ABCB5 on the cell surface. In culture, mesenchymal stem cells may be guided to differentiate into bone, fat, cartilage, or muscle cells using specific media. (Hirschi KK and, Goodell MA. Gene Ther. 2002; 9: 648-652. Pittenger MF, et al., Science. 1999; 284: 143-147. Schwartz RE, et al., J Clin Invest. 2002; 109: 1291- 1302. Hirschi K and Goodell M. Differentiation. 2001; 68: 186-192.)

The ABCB5 positive dermal mesenchymal stem cells can be obtained from skin. The skin may be derived from any subject having skin, but in some embodiments is preferably human skin. The skin may be derived from a subject of any age but in some embodiments is preferably adult skin, rather than adolescent or infant skin.

ABCB5⁺ cells have been identified as a phenotypically distinct dermal cell population able to provide immunoregulatory functions. Greater than 90% of ABCB5⁺ cells express MSC markers CD29, CD44, CD49e, CD73, CD105, and CD166, as well as the immune checkpoint receptor PD-1.

In other embodiments of the invention, ABCB5(+) stem cells are ocular stem cells. ABCB5(+) stem cells may be obtained from (e.g., isolated from or derived from) the basal limbal epithelium of the eye or from the retinal pigment epithelium (RPE). In some embodiments, ABCB5(+) stem cells are obtained from human eye. Other ABCB5(+) stem cell types such as, for example, those obtained from the central cornea may be used in various aspects and embodiments of the invention.

The cells of the invention also may possess multipotent differentiation capacity. In other words these cells not only define mesenchymal stromal cells (adipogenic, chondrogenic, osteogenic differentiation), but also other capacities, including differentiation to cells derived from of all three germ layers, i.e. 1. endoderm (e.g. angiogenesis - e.g. tube formation, CD31 and VEGFR1 expression), 2. mesoderm (e.g. myogenesis - e.g. spectrin, desmin expression) and 3. ectoderm (e.g. neurogenesis - e.g. Tujl expression).

In other embodiments of the invention, ABCB5(+) stem cells are synthetic stem cells. ABCB5+ stem cells isolated from human tissue can be passaged in culture to produce populations of cells that are structurally and functionally distinct from the original primary cells isolated from the tissue. These cells are referred to herein as synthetic or manufactured ABCB5+ stem cells. These cells are in vitro manufactured such that nearly all cells are in vitro progeny of physiologically occurring skin-derived ABCB5-positive mesenchymal stem cells that never existed in the context of the human body. Rather, they are newly created. The compositions of the invention are populations of cells. The term “population of cells” as used herein refers to a composition comprising at least two, e.g., two or more, e.g., more than one, synthetic ABCB5+ stem cells, and does not denote any level of purity or the presence or absence of other cell types, unless otherwise specified. In an exemplary embodiment, the population is substantially free of other cell types. In some embodiments greater than 99%, 99.5%, 99.7%, 99.9%, 99.99%, 99.998%, 99.999%, or 99.999997% of the population is an in vitro progeny of physiologically occurring skin-derived ABCB5-positive mesenchymal stem cells. The synthetic cells may also have distinct gene expression profiles relative to primary stem cells isolated from human tissue. The populations of synthetic cells (also referred to as ABCB5+ cells isolated from high passages) are different from the primary cells (those derived from low passage cultures that contain the native ABCB5+ cells found in the living organism). For example, certain stem cell markers are increased in high passage cells, e.g. SOX2, NANOG and SOX3, while certain mesenchymal stromal differentiation markers are decreased, e.g. MCAM, CRIG1 and ATXN1. The expression of selected sternness markers such as SSEA-4, DPP4 (CD26), PRDM1 (BLIMP1) and POU5F1 (OCT-4) in ABCB5+ cells in human skin at protein level was confirmed by immunostaining. While the expression of lower fibroblast lineage marker a-smooth muscle actin (a-SMA) was absent in ABCB5+ cells of human skin. These data support the finding that these late passage synthetic cells maintain pluripotent properties of ABCB5+ cells, and even have enhanced properties relative to the original cells.

In some preferred embodiments, 100% of the cells are synthetic, with 0% of the cells originating from the human tissue.

The ABCB5+ stem cells used herein are preferably isolated. An “isolated ABCB5+ stem cell” as used herein refers to a preparation of cells that are placed into conditions other than their natural environment. The term "isolated" does not preclude the later use of these cells thereafter in combinations or mixtures with other cells or in an in vivo environment.

The ABCB5+ stem cells may be prepared as substantially pure preparations.

The term “substantially pure” means that a preparation is substantially free of cells other than ABCB5 positive stem cells. For example, the ABCB5 cells should constitute at least 70 percent of the total cells present with greater percentages, e.g., at least 85, 90, 95 or 99 percent, being preferred. The cells may be packaged in a finished pharmaceutical container such as an injection vial, ampoule, or infusion bag along with any other components that may be desired, e.g., agents for preserving cells, or reducing bacterial growth. The composition should be in unit dosage form.

The ABCB5+ stem cells are useful for producing the CSCF compositions of the invention. In some embodiments the ABCB5+ stem cells may be administered to the subject to treat kidney disease, either alone or together with the CSCF compositions. In the embodiments when the ABCB5+stem cells are administered to a subject the cells may be autologous to the host (obtained from the same host) or non-autologous such as cells that are allogeneic or syngeneic to the host. Non-autologous cells are derived from someone other than the patient. Alternatively the ABCB5+stem cells can be obtained from a source that is xenogeneic to the host.

Allogeneic refers to cells that are genetically different although belonging to or obtained from the same species as the host or donor. Thus, an allogeneic human mesenchymal stem cell is a mesenchymal stem cell obtained from a human other than the intended recipient of the ABCB5+stem cells. Syngeneic refers to cells that are genetically identical or closely related and immunologically compatible to the host or donor, i.e., from individuals or tissues that have identical genotypes. Xenogeneic refers to cells derived or obtained from an organism of a different species than the host or donor.

When cells are administered an effective dose of cells should be given to a patient. The number of cells administered should generally be in the range of 1 x 10⁷ - 1 x 10¹⁰ and, in most cases should be between 1 x 10⁸ and 5 x 10⁹. Actual dosages and dosing schedules will be determined on a case by case basis by the attending physician using methods that are standard in the art of clinical medicine and taking into account factors such as the patient’s age, weight, and physical condition. The cells will usually be administered by intravenous injection or infusion although methods of implanting cells may be used as well.

The ABCB5+ stem cells are useful for producing a CSCF that is useful in the treatment of renal disease or kidney disease. In other embodiments ABCB5+ stem cells are useful for treating renal disease in a subject. As used herein, a subject is a human, non-human primate, cow, horse, pig, sheep, goat, dog, cat or rodent. Human synthetic ABCB5+stem cells and human subjects are particularly important embodiments.

Kidney disease is a common and expensive condition that is a major source of morbidity and mortality in humans. From a clinical perspective, kidney diseases can be classified as acute kidney injury (AKI) and chronic kidney disease (CKD). AKI, previously known called acute renal failure (ARF), is a clinical syndrome characterized by rapid deterioration of renal function that occurs within days. The principal feature of AKI is an abrupt decline in glomerular filtration rate (GFR), resulting in the retention of nitrogenous wastes (urea, creatinine).. To date, there is no specific treatment for established AKI.

AKI is characterized by an abrupt (within 48 hours) reduction in kidney function, which may include an absolute increase in serum creatinine of at least 0.3 mg/dl, a percentage increase in serum creatinine of at least 50%, or a reduction in urine output of less than 0.5 ml/kg per hour for more than six hours. Epidemiologically, AKI may be brought about post-renally, through obstruction of the urinary collection system by either intrinsic or extrinsic masses. Alternatively, AKI may originate within the renal system itself, by disorders of or injury affecting the structures of the nephron, such as the glomeruli, tubules, vessels, or interstitium.

CKD is a progressive loss of function over a prolonged period of time. An AKI patient who does not recover renal function may progress to CKD. Moreover, CKD- affected patients are prone to suffer AKI-like events.

The methods of the invention are useful for treating a subject having any of a number of kidney-related or renal disease states, for example, a subject having chronic kidney disease, end stage renal failure, diabetes, insulin resistance, kidney hypertrophy, kidney hypotrophy, polycystic kidney disease, proteinuria, hyperglycemia, hyperuricemia, uremic syndrome, gout, kidney stones, hypertension or hypertensive nephropathy, dyslipidemia, anemia and/or reduced erythropoietin production; iron deficiency or hyperfiltration, where such disease state is associated with a renal disorder, obstructive nephropathy, including one or more of the reduced or below normal glomerular filtration rate (GFR); elevated or above normal levels of serum creatinine (SCr); reduced or below normal levels of urine output; increased or above normal urinary excretion of neutrophil gelatinase-associated lipocalin (NGAL); signs of proteinuria (protein in the urine), such as albumin, 2 macroglobulin or IgG, in amounts greater than 3.5 g/day; hyperglycemia, hyperuricemia or dyslipidemia; anemia or reduced erythropoietin production; iron deficiency; or hyperfiltration.

The ABCB5+stem cells may be modified to express additional proteins which are also useful in the therapeutic indications, as described in more detail below. For example, the cells may include a nucleic acid that produces at least one bioactive factor which enhances ABCB5+stem cell activity and produces an altered CSCF. Thus, the ABCB5+stem cells may be genetically engineered (or transduced or transfected) with a gene of interest. The transduced cells can be administered to a patient in need thereof, for example to treat renal disease or may be cultured to produce enhanced CSCF which may be administered to treat disease.

The ABCB5+ stem cells, and progeny thereof, can be genetically altered. Genetic alteration of an ABCB5+ stem cell includes all transient and stable changes of the cellular genetic material which are created by the addition of exogenous genetic material. Exogenous genetic material includes nucleic acids or oligonucleotides, either natural or synthetic, that are introduced into the ABCB5+stem cells. The exogenous genetic material may be a copy of that which is naturally present in the cells, or it may not be naturally found in the cells. It typically is at least a portion of a naturally occurring gene which has been placed under operable control of a promoter in a vector construct.

Various techniques may be employed for introducing nucleic acids into cells. Such techniques include transfection of nucleic acid CaPO4 precipitates, transfection of nucleic acids associated with DEAE, transfection with a retrovirus including the nucleic acid of interest, liposome mediated transfection, and the like. For certain uses, it is preferred to target the nucleic acid to particular cells. In such instances, a vehicle used for delivering a nucleic acid according to the invention into a cell (e.g., a retrovirus, or other virus; a liposome) can have a targeting molecule attached thereto. For example, a molecule such as an antibody specific for a surface membrane protein on the target cell or a ligand for a receptor on the target cell can be bound to or incorporated within the nucleic acid delivery vehicle. For example, where liposomes are employed to deliver the nucleic acids of the invention, proteins which bind to a surface membrane protein associated with endocytosis may be incorporated into the liposome formulation for targeting and/or to facilitate uptake. Such proteins include proteins or fragments thereof tropic for a particular cell type, antibodies for proteins which undergo internalization in cycling, proteins that target intracellular localization and enhance intracellular half-life, and the like. Polymeric delivery systems also have been used successfully to deliver nucleic acids into cells, as is known by those skilled in the art. Such systems even permit oral delivery of nucleic acids.

One method of introducing exogenous genetic material into the ABCB5+stem cells is by transducing the cells using replication- deficient retroviruses. Replication- deficient retroviruses are capable of directing synthesis of all virion proteins, but are incapable of making infectious particles. Accordingly, these genetically altered retroviral vectors have general utility for high-efficiency transduction of genes in cultured cells. Retroviruses have been used extensively for transferring genetic material into cells. Standard protocols for producing replication-deficient retroviruses (including the steps of incorporation of exogenous genetic material into a plasmid, transfection of a packaging cell line with plasmid, production of recombinant retroviruses by the packaging cell line, collection of viral particles from tissue culture media, and infection of the target cells with the viral particles) are provided in the art.

A major advantage of using retroviruses is that the viruses insert efficiently a single copy of the gene encoding the therapeutic agent into the host cell genome, thereby permitting the exogenous genetic material to be passed on to the progeny of the cell when it divides. In addition, gene promoter sequences in the LTR region have been reported to enhance expression of an inserted coding sequence in a variety of cell types. The major disadvantages of using a retrovirus expression vector are (1) insertional mutagenesis, i.e., the insertion of the therapeutic gene into an undesirable position in the target cell genome which, for example, leads to unregulated cell growth and (2) the need for target cell proliferation in order for the therapeutic gene carried by the vector to be integrated into the target genome. Despite these apparent limitations, delivery of a therapeutically effective amount of a therapeutic agent via a retrovirus can be efficacious if the efficiency of transduction is high and/or the number of target cells available for transduction is high.

Yet another viral candidate useful as an expression vector for transformation of ABCB5+stem cells is the adenovirus, a double-stranded DNA virus. Like the retrovirus, the adenovirus genome is adaptable for use as an expression vector for gene transduction, i.e., by removing the genetic information that controls production of the virus itself. Because the adenovirus functions usually in an extrachromosomal fashion, the recombinant adenovirus does not have the theoretical problem of insertional mutagenesis. On the other hand, adenoviral transformation of a target mesenchymal stem cell may not result in stable transduction. However, more recently it has been reported that certain adenoviral sequences confer intrachromosomal integration specificity to carrier sequences, and thus result in a stable transduction of the exogenous genetic material.

Thus, as will be apparent to one of ordinary skill in the art, a variety of suitable vectors are available for transferring exogenous genetic material into dermal synthetic ABCB5+stem cells. The selection of an appropriate vector to deliver a therapeutic agent for a particular condition amenable to gene replacement therapy and the optimization of the conditions for insertion of the selected expression vector into the cell, are within the scope of one of ordinary skill in the art without the need for undue experimentation.

The promoter characteristically has a specific nucleotide sequence necessary to initiate transcription. Optionally, the exogenous genetic material further includes additional sequences (i.e., enhancers) required to obtain the desired gene transcription activity. For the purpose of this discussion an “enhancer” is simply any nontranslated DNA sequence which works contiguous with the coding sequence (in cis) to change the basal transcription level dictated by the promoter. Preferably, the exogenous genetic material is introduced into the dermal mesenchymal stem cell genome immediately downstream from the promoter so that the promoter and coding sequence are operatively linked so as to permit transcription of the coding sequence. A preferred expression vector includes an exogenous promoter element to control transcription of the inserted exogenous gene. Such exogenous promoters include both constitutive and inducible promoters.

Naturally-occurring constitutive promoters control the expression of essential cell functions. As a result, a gene under the control of a constitutive promoter is expressed under all conditions of cell growth. Exemplary constitutive promoters include the promoters for the following genes which encode certain constitutive or “housekeeping” functions: hypoxanthine phosphoribosyl transferase (HPRT), dihydrofolate reductase (DHFR) (Scharfmann et al., Proc. Natl. Acad. Sci. USA

88:4626-4630 (1991)), adenosine deaminase, phosphoglycerol kinase (PGK), pyruvate kinase, phosphoglycerol mutase, the actin promoter (Lai et al., Proc. Natl. Acad. Sci. USA 86: 10006-10010 (1989)), and other constitutive promoters known to those of skill in the art. In addition, many viral promoters function constitutively in eukaryotic cells. These include: the early and late promoters of SV40; the long terminal repeats (LTRS) of Moloney Leukemia Virus and other retroviruses; and the thymidine kinase promoter of Herpes Simplex Virus, among many others. Accordingly, any of the above- referenced constitutive promoters can be used to control transcription of a heterologous gene insert.

Genes that are under the control of inducible promoters are expressed only or to a greater degree, in the presence of an inducing agent, (e.g., transcription under control of the metallothionein promoter is greatly increased in presence of certain metal ions).

Inducible promoters include responsive elements (REs) which stimulate transcription when their inducing factors are bound. For example, there are REs for serum factors, steroid hormones, retinoic acid and cyclic AMP. Promoters containing a particular RE can be chosen in order to obtain an inducible response and in some cases, the RE itself may be attached to a different promoter, thereby conferring inducibility to the recombinant gene. Thus, by selecting the appropriate promoter (constitutive versus inducible; strong versus weak), it is possible to control both the existence and level of expression of a therapeutic agent in the genetically modified dermal mesenchymal stem cell. Selection and optimization of these factors for delivery of a therapeutically effective dose of a particular therapeutic agent is deemed to be within the scope of one of ordinary skill in the art without undue experimentation, taking into account the above- disclosed factors and the clinical profile of the subject.

In addition to at least one promoter and at least one heterologous nucleic acid encoding the therapeutic agent, the expression vector preferably includes a selection gene, for example, a neomycin resistance gene, for facilitating selection of

ABCB5+stem cells that have been transfected or transduced with the expression vector. Alternatively, the ABCB5+stem cells are transfected with two or more expression vectors, at least one vector containing the gene(s) encoding the therapeutic agent(s), the other vector containing a selection gene. The selection of a suitable promoter, enhancer, selection gene and/or signal sequence is deemed to be within the scope of one of ordinary skill in the art without undue experimentation.

The selection and optimization of a particular expression vector for expressing a specific gene product in an isolated stem cell is accomplished by obtaining the gene, preferably with one or more appropriate control regions (e.g., promoter, insertion sequence); preparing a vector construct comprising the vector into which is inserted the gene; transfecting or transducing cultured dermal synthetic ABCB5+stem cells in vitro with the vector construct; and determining whether the gene product is present in the cultured cells.

Thus, it is possible to genetically engineer ABCB5+stem cells in such a manner that, in addition to CSCF, they produce polypeptides, hormones and proteins not normally produced in human stem cells in biologically significant amounts or produced in small amounts but in situations in which overproduction would lead to a therapeutic benefit.

This invention is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings. The invention is capable of other embodiments and of being practiced or of being carried out in various ways. Also, the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of “including,” “comprising,” or “having,” “containing,” “involving,” and variations thereof herein, is meant to encompass the items listed thereafter and equivalents thereof as well as additional items.

EXAMPLES

Materials and methods

Animal experiments: All experiments were conducted in accordance with the German Animal Protection Law and approved by the local authority

(Regierungsprasidium Nordbaden, Karlsruhe Germany in agreement with EU guideline 2010/63/EU). Male SD rats (Janvier- Labs), about 200 g were used in all the experiments. The establishment of kidney failure and the assessment of the therapeutic potential of ABCB5+ cells and CM involved 24 and 49 animals respectively. 73 animals in total were used. Before the start of any experiment, animals were acclimatized for 1 week. The first 2 days following cisplatin administration, animals were housed in pairs in individually ventilated cages.

Transcutaneous assessment of renal function: The transcutaneous assessment of renal function was performed by using a transdermal device (MediBeacon GmbH, Mannheim, Germany). The optical part of the device consists of two light-emitting diodes, with an emission in the near infrared region (excitation: 706nm, emission: 790nm), and a photodiode detecting the fluorescent light. Energy is supplied by a small lithium polymer rechargeable battery with a voltage of 3.7V and capacity of 50 mAh.

The recorded digital data are stored in the internal memory of the device and are then downloaded onto a PC for the analysis. To perform the transcutaneous measurement of renal function, the device and the connected battery were attached to a specifically designed double-sided adhesive patch and then fixed on the shaved back of the animal.

The animal received a dose of 15mg/100 g BW of ABZWCY-HPβCD (stock solution 160 mg/ml in Deltajonin) via tail- vein injection, and its emitted fluorescent signal was recorded by the device. Shaving, fixation of the device and dose administration were performed under short isoflurane anesthesia (5% isoflurane at 3 L/min air flow, decreasing to 2% isoflurane at 1.5 L/min air flow). The measurement was performed for at least 2 hours, during which the animal was completely awake and freely moving. The recorded data represented the excretion curves of ABZWCY-HPβCD. To determine the half-life excretion of the administered ABZWCY-HPβCD, a 3-compartment model was applied using the open source freely-available software ‘GFRmeasure’ (https://www.mathworks.com/products/compiler/matlab-runtime.html).

Development of cisplatin-induced nephropathy model in SD rats: Kidney failure was induced by cisplatin administration. Cisplatin was purchased as a ready-to- use solution (TEVA, 1 mg/ml solution) and it was administered via ip route under short isoflurane anesthesia (5% isoflurane at 3 L/min air flow, decreasing to 2% isoflurane at 1.5 L/min air flow). To induce kidney damage, a single dose of 7 mg/kg BW was chosen.

Control animals received the same volume of sterile saline. 24 animals were used for the whole experiment (control n=3; cisplatin n=21). Six animals from the cisplatin group were excluded from the analysis because they died or were sacrificed before the end of the experiment. Therefore, 18 animals were finally included in the analysis (control n=3; cisplatin n=15, Table 1).

Table 1 Number of animals involved in the establishment of the cisplatin-induced nephrotoxicity model. Animals perfused on day 15 were included in the analysis The day of the first cisplatin/saline administration was considered as day 0. Evaluation days were performed 3 days before the start of the experiment (baseline), and 2, 7 and 14 days after cisplatin/saline administration. Animals were sacrificed on day 15 by perfusion (0.9% saline heparin 5 IU/mL pH=7 for 3min at 280mbar, PFA 4% for 3min at 230mbar) under anesthesia (Rompun 2% 5mg/Kg BW and Ketamin 10%,

100mg/Kg BW). The fresh left kidney was collected before perfusion and immediately snap-frozen in liquid nitrogen for subsequent mRNA extraction. Other organs (right kidney, spleen, liver, pancreas, intestine, heart, lungs) were collected after perfusion and processed for subsequent histology and immunofluorescence analyses. The experimental set-up is depicted in Fig. 1.

ABCB5+ cells and conditioned media: ABCB5+ cells and conditioned media were obtained from Ticeba-RHEACELL GmbH & Co. (Heidelberg, Germany). Conditioned media were obtained by collecting the supernatant of the following cell cultures:

CM: ABCB5+ cell culture;

CM+: ABCB5+ stimulated cell culture;

M+: human monocyte cell line THP-1 cultured with PMA to induce macrophage differentiation. After stimulation, macrophages polarize to Ml pro-inflammatory macrophages; coCM+: ABCB5+ co-cultured with THP-1 derived macrophages. After stimulation and in presence of ABCB5+ cells, THP-1 derived macrophages polarize to M2 anti-inflammatory macrophages.

Stimulation consisted of IFN-γ and LPS for promoting macrophage polarization. Cells were stimulated with 50IU/ml IFN-γ on the seeding day (first day) and with 50IU/ml IFN-γ + 20ng/ml LPS on the day after. The third day, conditioned media were harvested and immediately frozen and stored at -20C until injection into the animals or subsequent analysis. An overview of what the method is shown in Fig. 2.

Assessment of the therapeutic potential of human ABCB5+ cells and conditioned media in a cisplatin-induced nephropathy model: To assess the therapeutic potential of human ABCB5+ cells and conditioned media, the same experimental set-up used for the establishment of the cisplatin-induced nephrotoxicity model was used. Treatments were administrated on day 3. After baseline evaluation, on day 0 all animals received a single ip dose of cisplatin (7mg/kg BW). On day 2, blood and urine parameters were analyzed and the transcutaneous measurement of renal function was assessed in order to confirm whether the renal damage was properly induced. Thereafter, rats were randomly allocated to different groups according to the treatment received on day 3:

1) Group ivABCB5+: 2x10⁶ ABCB5+ cells iv administrated;

2) Group ipABCB5+: 2x10⁶ ABCB5+ cells ip administrated;

3) Group CM: 0.5ml of CM iv administrated;

4) Group CM+: 0.5ml of CM+ iv administrated;

5) Group coCM+: 0.5ml of coCM+ iv administrated;

6) Group vehicle: 0.5ml of fresh medium (RPMI1640 + L-Glutamine) iv administrated as a positive control.

The animals' health-status was evaluated on day 7 and 14, and the animals were sacrificed on day 15. The experimental set-up is depicted in Fig. 3.

49 animals were involved in the experiment (iv ABCB5+ n=12; ip ABCB5+ n=12; CM n=4; CM+ n=4, coCM n=12; vehicle n=5). Six animals (ip ABCB5+ n=1; CM n=1; CM+ n=1, coCM n=1; vehicle n=2) were excluded from the analysis because they died or were sacrificed before the end of the experiment. Therefore, 43 animals were finally included in the analysis (iv ABCB5+ n=12; ip ABCB5+n=11 ; CM n=3; CM+ n=3, coCM n=11 ; vehicle n=3, Table 2).

Statistical analysis: All statistical calculations regarding blood, urine and metabolic parameters as well as renal function values were performed using SAS JMP 13.0.0 (SAS Institute, Cary, NC, USA). Kruskal-Wallis tests were applied to evaluate the changes induced by cisplatin administration in the development of the model and the effect of the different therapies applied on injured animals. For all tests, nominal p- values <0.05 (*) or <0.005 (**) were considered as statistically significant.

Histology collection, staining, analysis and microscopy: On the day of the sacrifice, organs (right kidney, spleen, liver, pancreas, intestine, heart, lungs) were collected after perfusion and stored in 4% PFA for 24h. Fixed tissues were embedded in paraffin, cut (3μm) and stained with hematoxylin and eosin (H&E). H&E slices were used to detect morphological changes induced by cisplatin and full organ images were acquired using Axio Scan.Zl microscope (ZEISS).

RNA Isolation: For gene expression analysis, frozen left kidney samples obtained from sacrificed animals were used. Total RNA was extracted by using the RNeasy mini kit (Qiagen) following the manufacturer's instructions. The mRNA purity and integrity was tested by capillary electrophoresis on an Agilent 2100 bioanalyzer (Agilent) and high quality was confirmed. The isolated RNA was used for gene expression analysis.

Affymetrix GeneChips gene expression analysis and bioinformatics evaluation: Gene expression profiling was performed using arrays of human RatGene- 2_0-st-type from Affymetrix. From the RNA, biotinylated antisense cDNA was prepared according to the Affymetrix standard labelling protocol by using the GeneChip WT Plus Reagent Kit (Affymetrix, Santa Clara, USA) and the GeneChip Hybridization, Wash and Stain Kit (Affymetrix, Santa Clara, USA). The obtained cDNA was first hybridized on the chip with a GeneChip Hybridization oven 640, then dyed in the GeneChip

Fluidics Station 450 and thereafter scanned with a GeneChip Scanner 3000. All of the equipment used was from the Affymetrix-Company (Affymetrix, High Wycombe, UK). For array annotation, the Custom CDF Version 21 with ENTREZ based gene definitions was used. The raw fluorescence intensity values were normalized applying quantile normalization and RMA background correction. For identification of differentially expressed genes, OneWay-ANOVA was performed using the software package SAS JMP10 Genomics, version 6, from SAS (SAS Institute, Cary, NC, USA). A false positive rate of α= 0.05 with FDR correction was taken as the level of significance.

Gene Set Enrichment Analysis (GSEA) was performed by using the software R v3.4.0 (R Core Team 2017) and RStudio: Integrated development environment for R

(RStudio Boston, MA, USA). Pathways were obtained from Kyoto Encyclopedia of Genes and Genomes database (KEGG, http://www.genorne.ip/kegg). RNAseq gene and miRNAs expression and bioinformatics evaluation: New generation sequencing (NGS) gene expression profiling was performed with RNA sequencing (RNAseq) technology by BGI Tech Solutions Co. (Hong Kong) with BGISEQ-500 method. RNAseq data were analyzed and aligned by using RBioconducor and Rsubread software respectively. For annotation, the ENTREZ-based software package TxDb.Hsapiens.UCSC.hg19.knownGene was used and differential gene expression analysis was performed by a DESeq2 package. A false positive rate of α= 0.05 with FDR correction was taken as the level of significance. GSEA was performed by using the KEGG database version September 2018.

RNAseq technology also detects differentially expressed miRNA precursors. For miRNAs analysis, a false positive rate of α= 0.05 with FDR correction was taken as the level of significance. Putative target genes of differentially expressed miRNAs were predicted with the miRWalk program with a cutoff of binding p value=1. Putative target genes were compared with the differentially expressed genes found in the gene expression profiling. For functional interpretation of selected genes, the web-based online bioinformatics resource DAVID v6.8 (Database for Annotation, Visualization and Integrated Discovery) was used and BP-GO analysis was performed.

Cytokines assay: Simultaneous detection of 12 cytokines (IL2, IL4, IL5, IL6, IL10, IL12, IL13, IL17A, IFNy, TNFα, G-CSF, ΤGFβ1) in different conditioned media was assessed with the human Th1/Th2/Th17 cytokine Multi-Analyte ELISArray Kit (Qiagen) by following the manufacturer's instructions. Samples from 3 different donors were evaluated and the assay was repeated twice independently. Due to the kit range,

CM and CM+ were tested undiluted, while coCM+ and M+ were diluted 10 times. Results were obtained by using the infinite 200 PRO NanoQuant Microplate Readers (TECAN).

Example 1. Development and characterization of a cisplatin-induced nephrotoxicity model in SD rats.

A Cisplatin-induced nephrotoxicity model was established. Briefly, at the beginning of the experiment (day 0), a single intraperitoneal dose of cisplatin (7 mg/kg BW) was administrated to SD rats in order to induce renal injury. For monitoring the animal’s health state and for characterizing the induced damage, 3 days before (baseline) and 2, 7 and 14 days after cisplatin administration blood and urine were sampled for biochemical analyses, transcutaneous assessment of renal function was assessed and metabolic parameters were recorded. Kruskal- Wallis each paired comparison test of cisplatin-treated animals vs controls was applied and nominal p-values <0.05 (*) or <0.005 (**) were considered as statistically significant.

15 days after cisplatin administration, fresh left kidney was collected for gene expression analysis, while the other organs were collected after animal perfusion for histological evaluation.

2 days after cisplatin administration, both creatinine and urea increased by 1.6- fold and 2-fold respectively (creatinine p<0.005, urea p<0.05) compared to the corresponding controls and remained higher until day 14 (p<0.05). Particularly on day 7, we observed an increase of 7.6-fold for creatinine and 7.5-fold for urea (p<0.005).

Cisplatin also induced a 1.4-fold increase of plasma cholesterol already on day 2, which remained higher until day 14 (p<0.05) with respect to the control group. Additionally, the triglycerides levels on day 2 (p<0.05) in the cisplatin-treated animals, and then increased in the following days, by reaching a 1.2-fold increase on day 14.

Plasma electrolyte levels were also affected by cisplatin. Indeed, hypokalemia on day 2 (p<0.05) were reduced by half and hypophosphatemia on day 14 (p<0.005) was recorded in the treated animals compared to the controls. Hypercalcemia was also observed from day 7 onwards (p<0.05). In contrast, sodium level remained stable.

Plasma levels of liver enzymes were also measured. A reduction by half in AST levels was recorded on day 2 (p<0.05) in the treated animals compared to the controls. The AST value returned to basal levels on day 7. Additionally, GDHL levels increased by 2.5-fold on day 7 (not statistically significant) and halved on day 14. No differences in ALT and GGT levels were observed. No statistically significant differences among the groups were found for plasma levels of glucose and proteins.

The transcutaneous assessment of renal function was performed. ABZWCY- HPβCD half-life significantly increased by 1.8-fold already 2 days after cisplatin administration (p<0.05) and remained high until the end of the experiment, increasing by 2.1-fold on day 14. The observed changes in ABZWCY-HPβCD elimination curves represent the loss in renal function. H&E kidney sections have revealed profound changes in the renal cytoarchitecture due to cisplatin administration. The cortical region was the most affected area. Proximal tubules appeared highly degenerated with tubular cells detached from the basal membrane. Proximal tubule lumen appeared dilated and was often filled with protein casts or dregs of extruded necrotic cells. Around the tubules, fibrotic tissue deposition and inflammatory cell infiltration were detected. Due to the kidney's selfrecovery capacity, flat regenerating distal tubular cells were also present. These histological observations indicate that the kidney injury model was successfully established.

Example 2. Therapeutic effect of ABCB5+ cells and conditioned media in cisplatin- induced nephrotoxicity model in SD rats.

Three days after cisplatin administration, the therapeutic potential of ABCB5+ cells and different conditioned media was tested. The following experimental groups were created: ivABCB5+, ipABCB5+, CM, CM+, coCM and vehicle. For monitoring the state of health of the animals and for characterizing any possible effect due to the treatments, blood and urine were sampled for biochemical analyses, renal function was assessed and metabolic parameters were recorded 3 days before (baseline) and 2, 7 and 14 days after cisplatin administration. Kruskal-Wallis each paired comparison test of treated animals vs cisplatin-treated animals was applied and nominal p-values <0.05 (*) or <0.005 (**) were considered as statistically significant. 15 days after cisplatin administration, fresh left kidney was collected for gene expression analysis, while the other organs were collected after animal perfusion for histological evaluation.

The plasma biochemical results are summarized in Table 3 below.

On day 7, plasma creatinine decreased by 3.9 (p<0.05), 2.4 and 1.5 times in animals treated with CM, CM+ and co-CM+ respectively when compared to the cisplatin treated group, and remain low on day 14. Plasma urea decreased on day 7 by 3 and 1.8 times in animals treated with CM and CM+ respectively and remain low on day 14 when compared to the cisplatin treated group. No particular changes were found in animals who received ABCB5+ cells compared to cisplatin treated animals (Fig. 4).

Among the hepatic enzymes, GLDH levels decreased on day 7 by 3.7 (p<0.05) and 2.6 times in animals treated with CM and CM+ respectively. On day 14, GLDH levels were found significantly decreased by 1.3 times in co-CM+ treated animals compared to the cisplatin model. No changes were found in animals who received ABCB5+ cells compared to cisplatin treated animals (Fig. 5).

Plasma triglycerides significantly increase on day 7 (p<0.05) in animals treated with CM compared with the cisplatin treated group. No other significant changes of other plasma parameters were observed. Animals who received the vehicle did not show any particular differences in plasma parameters compared to the cisplatin-treated animals.

ABCB5+ cells/conditioned media were tested for their effect on urine parameters. The urine biochemistry results are summarized in Table 4.

Urine albumin levels decreased from day 7 onward in animals treated with all kind of conditioned media. On day 14, albuminuria decreased by 4.2, 2.1 and 1.8 times in animals treated with CM, CM+ and co-CM+ respectively when compared to the cisplatin treated group. No changes were found in animals who received ABCB5+ cells compared to the cisplatin treated animals Fig. 6. Only in CM treated animals, glycosuria significantly decreased on day 7 (p<0.05) and 14 (p<0.005) (Fig. 7). No other significant changes of other urine parameters were observed. Animals who received the vehicle did not show any particular differences in urine parameters compared to the cisplatin-treated animals.

The ABCB5+ cells/conditioned media were tested for their effect on renal function. Transcutaneous assessment of renal function was performed. The values of ABZWCY-HPβCD half-life are summarized in Table 5.

On day 7, animals treated with ABCB5+ cells and coCM+ showed a higher ABZWCY-HPβCD half-life when compared to the cisplatin treated animals (p<0.05), while the CM+ group had a 1.8-times smaller ABZWCY-HPβCD half-life. One week later, animals treated with the 3 different conditioned media had a similar ABZWCY- HPβCD excretion time, which was lower than in the control, while animals treated with ABCB5+ cells maintained a high ABZWCY-HPβCD half-life (Fig. 8).

It was demonstrated that ABCB5+ cells/conditioned media effect on body weight, diuresis, food and water intake. Before and after being in metabolic cages for 16 hours, changes in diuresis, BW, food and water intake were recorded. The values are summarized in Table 6.

Loss of weight gain, in a range of 20-30 g, was recorded between day 2 and 7 in animals who received ABCB5+ cells and coCM+. Animals who received CM and CM+ gained weight or maintained a quite stable weight (CM p<0.05). Accordingly, in these groups the food intake, which decreased on day 2, was higher than in the other animal groups (CM p<0.05).

The impact of ABCB5+ cells/conditioned media on renal morphology was tested. Whole kidney scans were examined and showed changes in the renal cytoarchitecture of animals treated with ABCB5+ cells and conditioned media. Animals that received ABCB5+ cells, both ip and iv, did not show any visible differences from the cisplatin treated animals. The cortical and juxtamedullary region was the most affected area with dilated proximal tubuli in the cortical region and protein cast accumulations mostly in the papillary region. Animals treated with conditioned media showed less severe damage in terms of affected area and protein cast accumulations. CM treated animals resulted to be affected mainly in the juxtamedullary region.

Example 3. Gene expression and miR expression resulting from exposure to ABCB5+ cells and conditioned media in cisplatin-induced nephrotoxicity model in SD rats.

Gene expression analysis of renal tissue obtained from sacrificed animals was performed. The analysis was first performed with both microarray and RNAseq, showing reliable results with both approaches. However, due to its innovative and more sensitive methodology, and for the possibility to additionally perform miRNAs profiling, RNAseq was chosen over microarray for subsequent analyses and is referred to as a gene set enrichment analysis (GSEA).

The GSEA analysis showed 49 significant differentially expressed pathways. Cisplatin led to upregulation of apoptosis and p53 signaling pathways. The renal tissue metabolism is downregulated and so the translational activity and protein processing. In contrast, upregulation of DNA replication, cell cycle, ribosome and spliceosome assembly, and of several pathways involved in extracellular matrix (ECM) remodeling was found. Additionally, upregulation of immune system and signal transduction pathways were observed.

The effect of ABCB5+ cells/conditioned media effect on gene expression: RNAseq analysis was tested. Cluster analysis of 21 RNAseq samples showed that, among the seven experimental groups, two distinct clusters could be distinguished. Control animals and 2 over 3 animals treated with CM and CM+ clustered together, while all animals treated with ABCB5+ cells and coCM+ bunched with the cisplatin- treated animals in a heatmap analysis.

For GSEA analysis, pairwise comparisons of each treatment group to the cisplatin were performed. A total of 126 significant (adj p-value< 0.05) differently expressed pathways were found. For some pathways, a different trend of expression in the different experimental groups was observed. An overview of up and down regulated pathways in the different groups is shown in Fig. 9. The number values are presented in Table 7. Upregulation of xenobiotic degradation and metabolism pathways were found in the animals treated with any therapy. Overall, upregulation of several metabolic pathways was mainly found in the animals treated with CM and coCM+ (Table 7).

1.5; dark red: NES >2).

Translation, proteasome and ribosome biogenesis are unregulated in animals treated with ABCB5 cells and coCM+. Additionally, the DNA replication and reparation activity is upregulated in ABCB5 cells groups. In contrast, these pathways are downregulated in the CM and CM+ groups (Table 8).

A strong downregulation of signaling pathways was observed in the animals treated with ipABCB56+, ivABCB5+ and coCM+, while no significant changes were found in the CM and CM+ groups. Interestingly, the dowmegulation of the cytokine- cytokine receptor interaction pathway was found in all groups. Dowmegulation of pathways involved in the immune system was mainly observed in the ipABCB5+, coCM+ and CM groups. Pathways involved in the regulation of actin cytoskeleton and cellular junctions were found downregulated in ipABCB5+, ivABCB5+ and coCM+ (Table 9).

In the cisplatin treated animals, the expression of 14 miRNAs was found significantly changed (adj. p<0.05); 6 miRNAs were downregulated, whereas 8 were upregulated. The top five upregulated (mir-5132, mir-1199, mir-196b, mir-6321, mir- 10a) and downregulated (mir-3120, mir-155, mir-214, mir-142, mir-147) miRNAs were selected for the subsequent analysis. By using the miRWalk program with a cutoff of binding p value=1, the putative target genes of the 10 aforementioned miRNAs were predicted.

The analysis showed 6192 and 5196 target genes of the five top downregulated and upregulated miRNAs respectively. The putative genes were compared with the 6333 significant (adj. p<0.05) differentially expressed genes found in the RNAseq analysis. Genes in common between the two groups were selected. As a result, 2185 and 1898 overlapping genes of the downregulated and upregulated miRNAs respectively were selected and used for the further analysis.

ABCB5+ cells and derived conditioned media were tested for an effect on miRNA expression. Both were found to affect miRNAs expression. Table 10 illustrates the differentially expressed miRNAs found in the different groups. 6 differentially expressed miRNAs were found in ipABCB5+ treated animals (downregulated: miR-7a- 1, miR-632; upregulated: miR-3562, miR-3596b, miR-let-7d, miR-3594), 6 in CM+ (downregulated: miR-147, miR-155, miR-7a-l; upregulated: miR-5132, miR-196b, miR- 3594), 2 in CM (upregulated: miR-5132, miR-186) and 1 in coCM+ (downregulated: miR-142) and ivABCB5+ (upregulated: miR-6318). Five over 14 differentially expressed miRNAs found in the cisplatin-induced nephrotoxicity model were found oppositely regulated in animals treated with CM (miR-5132), coCM+ (miR-142) and CM+ (miR-5132, miR-196b, miR-147, miR-155).

We checked for putative target genes of the miRNAs and we selected only the ones that overlaps with the differentially expressed genes found in each experimental group by RNAseq gene expression profiling. As result, only upregulated miRNAs found in CM treated animals showed a sufficient number of selected target genes to perform BP-GO analysis, which was then carried out using the DAVID database. The obtained BP-GO terms are reported in Table 11.

Example 4. Cytokine Analysis resulting from exposure to ABCB5+ cells and conditioned media in cisplatin-induced nephrotoxicity model in SD rats.

By using an ELISA array, conditioned media were tested for the following 12 cytokines: IL2, IL4, IL5, IL6, IL10, IL12, IL13, IL17A, ΙFΝγ, TNFα, G-CSF, ΤGFβ1. The result showed changes among the groups in ΤGFβ1, TNFα, IL6 and G-CSF levels. IL6 and G-CSF levels were higher in coCM+ compared to the other conditioned media. Higher levels of ΤGFβ1 and TNFα were found in M+ when compared to the other conditioned media (Fig. 10).

All references cited herein are fully incorporated by reference. Having thus described several aspects of at least one embodiment of this invention, it is to be appreciated various alterations, modifications, and improvements will readily occur to those skilled in the art. Such alterations, modifications, and improvements are intended to be part of this disclosure, and are intended to be within the spirit and scope of the invention. Accordingly, the foregoing description and drawings are by way of example only.

Claims

CLAIMS What is claimed is:

1. A composition, comprising a secreted stem cell factor (CSCF), wherein the CSCF is isolated from a population of ABCB5+ stem cells.

2. The composition of claim 1, wherein the CSCF is present in an ABCB5+ cell derived conditioned medium.

3. The composition of claim 2, wherein the ABCB5+ cell derived conditioned medium is isolated from a population of ABCB5+ stem cells co-cultured with macrophage.

4. The composition of claim 2 or 3, wherein the ABCB5+ cell derived conditioned medium is isolated from a population of ABCB5+ stem cells stimulated with IFN-γ.

5. The composition of any one of claims 2-4, wherein the ABCB5+ cell derived conditioned medium is isolated from a population of ABCB5+ stem cells stimulated with lipopolysaccharide (LPS).

6. The composition of any one of claims 1-5, wherein the ABCB5+ stem cells are dermal mesenchymal stem cells.

7. The composition of any one of claims 1-5, wherein the ABCB5+ stem cells are timbal stem cells.

8. The composition of any one of claims 1-5, wherein the ABCB5+ stem cells are ocular stem cells.

9. The composition of any one of claims 1-8, wherein the CSCF is conditioned medium.

10. The composition of any one of claims 1-8, wherein the CSCF is selected from the group consisting of a polypeptide, a nucleic acid, a small molecule and an exosome.

11. A method for preparing a composition of a secreted stem cell factor

(CSCF), comprising, culturing a population of ABCB5+ stem cells for at least two days after the cells are added to a dish for culturing (seeding day), isolating a culture medium from the cell population, and formulating the isolated culture medium as a composition of a CSCF.

12. The method of claim 11, further comprising purifying the CSCF from the isolated culture medium.

13. The method of claim 11 or 12, wherein the ABCB5+ stem cells are co- cultured with macrophage.

14. The method of claim 13, wherein the macrophage is a human monocyte cell line THP-1 cultured with PM A to induce macrophage differentiation and polarized to form Ml pro-inflammatory macrophages.

15. The method of any one of claims 11-14, wherein the ABCB5+ stem cells are stimulated with IFN-γ.

16. The method of claim 15, wherein 50IU/ml IFN-γ is added to the ABCB5+ stem cells on the seeding day.

17. The method of any one of claims 11-16, wherein the ABCB5+ stem cells are stimulated with lipopolysaccharide (LPS).

18. The method of claim 17, wherein 50IU/ml IFN-γ and 20ng/ml LPS is added to the ABCB5+ stem cells one day after the seeding day.

19. The method of any one of claims 11-18, wherein the ABCB5+ stem cells are cultured for three days following the seeding day and the culture medium is isolated on the third day.

20. The method of any one of claims 11-18, wherein the conditioned medium is formulated for administration to a subject without further purification.

21. The method of any one of claims 11-18, wherein the conditioned medium is further processed to separate the CSCF from other components.

22. The method of any one of claims 11-18, wherein the CSCF is isolated and purified from the conditioned medium.

23. The method of claim 22, wherein the CSCF is a polypeptide

24. The method of claim 23, wherein the polypeptide comprises a cytokine.

25. The method of claim 22, wherein the CSCF is an exosome.

26. A method for treating renal disease in a inducing liver tissue generation in a subject in need thereof, comprising injecting a composition comprising a secreted stem cell factor (CSCF), wherein the CSCF is isolated from a population of ABCB5+ stem cells into the subject in an effective amount to treat renal disease.

27. The method of claim 26, wherein the composition is a composition of any one of claims 1-10.

28. The method of claim 26, wherein the composition is a composition made according to a method of any one of claims 11-25.

29. The method of any one of claims 26-28, wherein the composition is administered systemically into the subject by intravenous or intraperitoneal delivery.