WO2024133886A1

WO2024133886A1 - Primed mesenchymal stem cells for use in the treatment of chronic kidney disease

Info

Publication number: WO2024133886A1
Application number: PCT/EP2023/087606
Authority: WO
Inventors: Jan H. SPAAS; Eva Depuydt; Glenn PAUWELYN
Original assignee: Boehringer Ingelheim Veterinary Medicine Belgium
Priority date: 2022-12-23
Filing date: 2023-12-22
Publication date: 2024-06-27

Abstract

The current invention relates to primed mesenchymal stem cells (MSCs) or a pharmaceutical composition comprising a therapeutically effective amount of said primed MSCs for use in the treatment of chronic kidney disease in an affected feline, wherein said composition is administered to an affected feline and wherein prior to said administration the MSCs are primed by in vitro culturing said MSCs in an inducing cell medium comprising TNF-α. The invention further relates to a pharmaceutical composition comprising peripheral blood-derived MSCs, to TNF-α primed MSCs and renal primed MSCs or a composition comprising said primed MSC.

Description

PRIMED MESENCHYMAL STEM CELLS FOR USE IN THE TREATMENT OF CHRONIC KIDNEY DISEASE

FIELD OF THE INVENTION

The present invention relates to primed mesenchymal stem cells for use in the treatment of chronic kidney disease in felines.

BACKGROUND

Chronic kidney disease (CKD) is a common medical condition in geriatric cats and is characterized histologically by tubulointerstitial inflammation and fibrosis, with subsequent progressive loss of renal function. Healthy kidneys perform many important functions, most notably filtering the blood and making urine to excrete waste products, maintaining fluid balance in the body, producing certain hormones, and regulating electrolytes. In CKD, all these regulatory processes can be interfered with, and can therefore result in a variety of health problems for an animal. Currently, renal transplant is the only definitive therapy to improve kidney function in cats with CKD. Therefore, novel and effective therapeutic options are highly sought after to provide additional treatment options for cats suffering from this disease. To date, no known treatments that stop disease progression or repair affected kidneys have been identified.

Mesenchymal stem cells (MSCs) are being explored as a treatment for CKD in both people and animals, because of their capability to modulate inflammatory responses and mediate cell-cell interactions to promote tissue repair. Several animal studies have investigated their safety and efficacy in treatment and showed interesting results. A study in eight CKD cats however suggested that allogeneic MSCs from cryopreserved adipose tissue had no improvement after administration.

There thus remains a need in the art for an improved use of MSCs to slow down the disease progression and/or even reverse the pathological condition of chronic kidney disease in the family of cats. The present invention targets at solving at least one of the aforementioned disadvantages.

SUMMARY OF THE INVENTION The present invention and embodiments thereof serve to provide a solution to one or more of above-mentioned disadvantages. To this end, the present invention relates to primed mesenchymal stem cells (MSCs) or a pharmaceutical composition comprising a therapeutically effective amount of said primed MSCs for use in the treatment of chronic kidney disease in an affected feline according to claim 1. More particular, the invention relates to primed MSCs or a pharmaceutical composition comprising a therapeutically effective amount of said primed MSCs for use in the treatment of chronic kidney disease in an affected feline wherein said composition is administered to an affected feline and wherein prior to said administration the MSCs are primed by in vitro culturing said MSCs in an inducing cell medium comprising TNF-o.

In embodiments, said inducing cell medium further comprises interleukin IL-ip. In embodiments, said MSCs are intravenously administered. In embodiments, said MSCs being administered are xenogeneic MSCs.

In a second aspect, the present invention relates to a pharmaceutical composition comprising peripheral blood-derived MSCs according to claim 14. More particular, the invention relates to a pharmaceutical composition comprising peripheral blood- derived MSCs, wherein said MSCs are mammal-derived, preferably equine-derived, and present in a sterile liquid at a concentration of between 10⁵ - 10⁷ MSCs per mL of said composition, wherein said composition has a volume of about 0.5 to 5 ml, wherein said MSCs measure positive for mesenchymal markers CD29, CD44 and CD90 and measure negative for MHC class II molecules and CD45, wherein said MSCs have a suspension diameter between 10 pm and 100 pm and wherein said MSCs are primed by means of an in vitro culturing step in an inducing cell medium comprising TNF-o. In an embodiment, said inducing cell medium further comprises interleukin (IL)-ip.

In further aspects, the present invention relates to TNF-o primed MSCs and a composition comprising renal primed MSCs, wherein said MSCs are preferably equine-derived and derived from blood, preferably peripheral blood.

The inventors surprisingly found that by using said primed MSCs the disease could be more efficiently treated compared to the use of unprimed MSCs in an affected feline suffering from chronic kidney disease. This was for instance evidenced by an increased quality of life, a more pronounced decrease in blood pressure values and positive effects on the body weight of the treated animals. DESCRIPTION OF FIGURES

The following description of the figures of specific embodiments of the invention is merely exemplary in nature and is not intended to limit the present teachings, their application or uses. Throughout the drawings, corresponding reference numerals indicate like or corresponding parts and features.

Figure 1 shows PGE₂ secretion data of primed equine peripheral blood derived mesenchymal stem cells (ePB-MSCs) according to an embodiment of the present invention, represented as fold change compared to PGE₂ secretion of unprimed ePB- MSCs.

Figure 2 shows IL-6 secretion data of unprimed equine peripheral blood derived mesenchymal stem cells (ePB-MSCs) and primed ePB-MSCs according to an embodiment of the present invention.

Figure 3 shows Quality of life (QoL) scores, normalized to baseline values obtained on Day 0, in 18 cats suffering from chronic kidney disease stage 2 or 3. Seven cats were treated with native ePB-MSCs, six cats were treated with primed ePB-MSCs according to an embodiment of the invention and five cats were treated with placebo (saline).

Figure 4 shows systolic blood pressure values (mmHg), normalized to baseline values obtained on Day 0, in 18 cats suffering from chronic kidney disease stage 2 or 3. Seven cats were treated with native ePB-MSCs, six cats were treated with primed ePB-MSCs according to an embodiment of the invention and five cats were treated with placebo (saline).

Figure 5 shows Body weight (kg), normalized to baseline values obtained on Day 0, in 18 cats suffering from chronic kidney disease stage 2 or 3. Seven cats were treated with native ePB-MSCs, six cats were treated with primed ePB-MSCs according to an embodiment of the invention and five cats were treated with placebo (saline).

DETAILED DESCRIPTION OF THE INVENTION

The present invention concerns primed MSCs or a pharmaceutical composition comprising a therapeutically effective amount of said primed MSCs for use in the treatment of chronic kidney disease in an affected feline. Priming of said MSCs occurs by in vitro culturing said MSCs in an inducing cell medium comprising TNF- o. In a preferred embodiment, said inducing cell medium further comprises interleukin IL-ip.

The inventors surprisingly found that by using said primed MSCs the disease could be more efficiently treated compared to the use of unprimed MSCs in an affected feline suffering from chronic kidney disease. This was for instance evidenced by an increased quality of life, a more pronounced decrease in blood pressure values and positive effects on the body weight of the treated animals.

Unless otherwise defined, all terms used in disclosing the invention, including technical and scientific terms, have the meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. By means of further guidance, term definitions are included to better appreciate the teaching of the present invention.

As used herein, the following terms have the following meanings:

"A", "an", and "the" as used herein refers to both singular and plural referents unless the context clearly dictates otherwise. By way of example, "a compartment" refers to one or more than one compartment.

"About" as used herein referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, is meant to encompass variations of +/- 20% or less, preferably +/-10% or less, more preferably +/-5% or less, even more preferably +/-1% or less, and still more preferably +/-0.1% or less of and from the specified value, in so far such variations are appropriate to perform in the disclosed invention. However, it is to be understood that the value to which the modifier "about" refers is itself also specifically disclosed.

"Comprise", "comprising", and "comprises" and "comprised of" as used herein are synonymous with "include", "including", "includes" or "contain", "containing", "contains" and are inclusive or open-ended terms that specifies the presence of what follows e.g. component and do not exclude or preclude the presence of additional, non-recited components, features, element, members, steps, known in the art or disclosed therein. Furthermore, the terms first, second, third and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order, unless specified. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein.

The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within that range, as well as the recited endpoints.

The expression "% by weight", "weight percent", "%wt" or "wt%", here and throughout the description unless otherwise defined, refers to the relative weight of the respective component based on the overall weight of the formulation.

Whereas the terms "one or more" or "at least one", such as one or more or at least one member(s) of a group of members, is clear per se, by means of further exemplification, the term encompasses inter alia a reference to any one of said members, or to any two or more of said members, such as, e.g., any >3, >4, >5, >6 or >7 etc. of said members, and up to all said members.

Unless otherwise defined, all terms used in disclosing the invention, including technical and scientific terms, have the meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. By means of further guidance, definitions for the terms used in the description are included to better appreciate the teaching of the present invention. The terms or definitions used herein are provided solely to aid in the understanding of the invention.

Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment, but may. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner, as would be apparent to a person skilled in the art from this disclosure, in one or more embodiments. Furthermore, while some embodiments described herein include some but not other features included in other embodiments, combinations of features of different embodiments are meant to be within the scope of the invention, and form different embodiments, as would be understood by those in the art. For example, in the following claims, any of the claimed embodiments can be used in any combination.

The terms "mesenchymal stem cells", "MSCs" or "mesenchymal stromal cells" refer to multipotent, self-renewing cells that express a specific set of surface antigens and can differentiate into various cell types. MSCs can secrete many different cytokines and growth factors, which regulate immune activity and enhance the potential of expansion and differentiation of host cells, thus promoting the recovery of damaged tissues.

The term "isolated", refers to both the physical identification and isolation of cells from a cell culture or a biological sample, like blood, that can be performed by applying appropriate cell biology technologies that are either based on the inspection of cell cultures and on the characterization (and physical separation when possible and desired) of cells corresponding to the criteria, or on the automated sorting of cells according to the presence/absence of antigens and/or cell size (such as by FACS). In some embodiments, the terms "isolating" or "isolation" may comprise a further step of physical separation and/or quantification of the cells, especially by carrying out flow cytometry.

The term "in vitro" as used herein denotes outside, or external to, a body. The term "in vitro" as used herein should be understood to include "ex vivo". The term "ex vivo" typically refers to tissues or cells removed from a body and maintained or propagated outside the body, e.g., in a culture vessel or a bioreactor.

The term "passage" or "passaging" is common in the art and refers to detaching and dissociating the cultured (mesenchymal stem) cells from the culture substrate and from each other. For sake of simplicity, the passage performed after the first time of growing the cells under adherent culture conditions is generally referred to as "first passage" (or passage 1, Pl). The cells may be passaged at least one time and preferably two or more times. Each passage subsequent to passage 1 is referred to with a number increasing by 1, e.g., passage 2, 3, 4, 5, or Pl, P2, P3, P4, P5, etc.

The term "cell medium" or "cell culture medium" or "medium" refers to an aqueous liquid or gelatinous substance comprising nutrients which can be used for maintenance or growth of cells. Cell culture media can contain serum or be serum- free. The cell medium may comprise or be supplemented with growth factors. The term "growth factor" as used herein refers to a biologically active substance which influences proliferation, growth, differentiation, survival and/or migration of various cell types, and may effect developmental, morphological and functional changes in an organism, either alone or when modulated by other substances. A growth factor may typically act by binding, as a ligand, to a receptor (e.g., surface or intracellular receptor) present in cells.

"Autologous" administration of MSCs in the present context refers to MSCs from a donor being administered to a recipient, wherein both recipient and donor are the same.

"Allogeneic" administration of MSCs in the present context refers to MSCs from a donor being administered to a recipient, wherein both recipient and donor are of the same species, but are not the same.

"Xenogeneic" administration of MSCs in the present context refers to MSCs from a donor being administered to a recipient, wherein the recipient and the donor are from different species.

"Native MSCs" in the context of the present invention refers to MSCs which have not been exposed to a stimuli environment, such as inflammatory mediators.

As used herein, the "inflammatory environment" or "inflammatory condition" refers to a state or condition characterized by (i) an increase of at least one pro- inflammatory immune cell, pro-inflammatory cytokine, or pro-inflammatory chemokine; and (ii) a decrease of at least one anti-inflammatory immune cell, antiinflammatory cytokine, or anti-inflammatory chemokine.

The term "anti-inflammatory", "anti-inflammation", "immunosuppressive", and "immunosuppressant" refers to any state or condition characterized by a decrease of at least one indication of localized inflammation (such as, but not limited to, heat, pain, swelling, redness, and loss of function) and/or a change in systemic state characterized by (i) a decrease of at least one pro-inflammatory immune cell, pro- inflammatory cytokine, or pro-inflammatory chemokine; and (ii) an increase of at least one anti-inflammatory immune cell, anti-inflammatory cytokine, or antiinflammatory chemokine. The "population doubling time" or "PDT" of current invention is to be calculated by the formula: PDT = T/(ln(Nf/Nj)/ln(2)), whereby T is the cell culture time (in days) to reach 80% confluency, Nf is the final number of cells after cell detachment and whereby Nj is the initial number of cells at time point zero.

By the term "anti-coagulant", it is meant a composition that can inhibit the coagulation of the blood. Examples of anticoagulants used in the present invention include EDTA or heparin.

The term "buffy coat" in this invention, is to be understood as the fraction of noncoagulated blood, preferably obtained by means of a density gradient centrifugation, whereby the fraction is enriched with white blood cells and platelets.

The term "blood-inter-phase" is to be understood as that fraction of the blood, preferably obtained by means of a density gradient, located between the bottom fraction, mainly consisting of erythrocytes and polymorphonuclear cells, and the upper fraction, mainly consisting of plasma. The blood-interphase is the source of blood mononuclear cells (BMCs) comprising monocytes, lymphocytes, and MSCs.

The term "suspension diameter" as used herein, is understood as the mean diameter of the cells, when being in suspension. Methods of measuring diameters are known in the art. Possible methods are flow cytometry, confocal microscopy, image cytometer, or other methods known in the art.

The term "therapeutically effective amount" is the minimum amount or concentration of a compound or composition that is effective to reduce the symptoms or to ameliorate the condition of a disease.

The term "treatment" refers to both therapeutic, prophylactic or preventive measures to reduce or prevent pathological conditions or disorders from developing or progressing. In the context of the present invention, "treatment" preferably refers to "therapeutic treatment", wherein the primed mesenchymal stem cells (MSCs) or a pharmaceutical composition comprising a therapeutically effective amount of said primed MSCs are administered to a feline suffering from chronic kidney disease.

"Chronic kidney disease" (CKD) is a type of kidney disease in which there is gradual loss of kidney function over a period of months to years. Chronic renal disease (CRD), chronic renal failure (CRF), and chronic renal insufficiency refer to the same condition. For animals such as cats, typical visual signs of CKD may include lethargy, weight loss, urinating greater volumes and drinking more water to compensate due to the loss of the ability to concentrate their urine appropriately, loss of appetite, elevated blood pressure (hypertension) affecting eyes, brain and/or heart, and/or pale gums due to a reduction of red blood cells (anemia).

Following diagnosis of CDK, staging of CDK may be undertaken to facilitate appropriate treatment and monitoring of the patient. "IRIS (International Renal Interest Society) stages" are determined based initially on fasting blood creatinine concentration assessed on at least two occasions in a hydrated, stable patient. At stage 1, the patient has a normal blood creatinine level, and at the final stage 4, the patient has increasing risk of systemic clinical signs and uremic crises (see Table 1 below). Table 1: IRIS stages of chronic kidney disease in cats

The terms "patient", "subject", "animal", or "mammal" are used interchangeably and refer to a mammalian subject to be treated. Preferably, the mammal is a feline, such as a cat.

"Feline" or "felines" in the present invention refers to cats of the Felidae family. A member of this family is also called a felid. The living Felidae are divided in two subfamilies: the Pantherinae and Felinae. Pantherinae includes five Panthera and two Neofelis species, while Felinae includes the other 34 species in ten genera, amongst which domestic cats, cheetahs, servals, lynx' and cougars.

"Priming" or "cell priming" (also referred to as "licensing" or "preconditioning") in the present invention consists of preparing cells for some specific function or lineage-specific differentiation, which involves cell activation, molecular signaling, genetic or epigenetic modifications, and morphology/phenotype changes. This concept is commonly used in the immunology field, and it has been adapted for the stem cell scope. Several priming approaches have been proposed in the last years to improve MSC function, survival, and therapeutic efficacy. These priming approaches can be divided into five categories: (a) MSC priming with inflammatory cytokines or mediators, (b) MSC priming with hypoxia, (c) MSC priming with pharmacological drugs and chemical agents, (d) MSC priming with biomaterials and different culture conditions, and (e) MSC priming with other molecules. In the present invention, "priming" is used in the context of the first category ("MSC priming with inflammatory cytokines or mediators"). MSCs primed with inflammatory cytokines or mediators have a different expression and/or secretion of anti-inflammatory and immunomodulatory factors. Such priming with inflammatory cytokines or mediators often occurs by in vitro culturing said MSC in an inducing cell medium comprising the specific inflammatory cytokine(s) or mediator(s).

"Inducing cell medium" in the present invention refers to a medium appropriate for culturing cells and further comprising one or more inflammatory cytokine(s) or mediator(s) for priming the cells. Such "inducing cell medium" can also be referred to as a "cell priming medium". Media appropriate for cell culture are known in the cell culturing field and are further discussed below. For example, a pro-inflammatory cytokine may be added to a basal cell medium during MSC culture to augment the anti-inflammatory effects of the MSCs. In an embodiment, the inducing cell medium comprises Dulbecco's Modified Eagle's Medium (DMEM) to which TNF-o is added, wherein TNF-o is present in said inducing cell medium in a concentration between 1 ng/ml to 50 ng/ml, preferably between 5 ng/ml and 15 ng/ml (such as 5 ng/ml, 6 ng/ml, 7 ng/ml, 8 ng/ml, 9 ng/ml, 10 ng/ml, 11 ng/ml, 12 ng/ml, 13 ng/ml, 14 ng/ml or 15 ng/ml). In an alternative embodiment, the inducing cell medium comprises DMEM to which TNF-o and IL-1 p are added, wherein TNF-o is present in said inducing cell medium in a concentration between 1 ng/ml to 50 ng/ml, preferably between 5 ng/ml and 15 ng/ml (such as 5 ng/ml, 6 ng/ml, 7 ng/ml, 8 ng/ml, 9 ng/ml, 10 ng/ml, 11 ng/ml, 12 ng/ml, 13 ng/ml, 14 ng/ml or 15 ng/ml) and wherein IL-ip is present in said inducing cell medium in a concentration between 1 ng/ml to 50 ng/ml, preferably between 5 ng/ml and 15 ng/ml (such as 5 ng/ml, 6 ng/ml, 7 ng/ml, 8 ng/ml, 9 ng/ml, 10 ng/ml, 11 ng/ml, 12 ng/ml, 13 ng/ml, 14 ng/ml or 15 ng/ml).

In a preferred embodiment, the inducing cell medium comprises Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 5-10% FBS (such as 5% FBS, 6% FBS, 7% FBS, 8% FBS, 9% FBS or 10% FBS) to which TNF-o is added, and wherein TNF- o is present in said inducing cell medium in a concentration between 1 ng/ml to 50 ng/ml, preferably between 5 ng/ml and 15 ng/ml (such as 5 ng/ml, 6 ng/ml, 7 ng/ml, 8 ng/ml, 9 ng/ml, 10 ng/ml, 11 ng/ml, 12 ng/ml, 13 ng/ml, 14 ng/ml or 15 ng/ml). In an alternative embodiment, the inducing cell medium comprises DMEM supplemented with 5-10% FBS (such as 5% FBS, 6% FBS, 7% FBS, 8% FBS, 9% FBS or 10% FBS), to which TNF-o and IL-13 are added, wherein TNF-o is present in said inducing cell medium in a concentration between 1 ng/ml to 50 ng/ml, preferably between 5 ng/ml and 15 ng/ml (such as 5 ng/ml, 6 ng/ml, 7 ng/ml, 8 ng/ml, 9 ng/ml, 10 ng/ml, 11 ng/ml, 12 ng/ml, 13 ng/ml, 14 ng/ml or 15 ng/ml) and wherein IL-13 is present in said inducing cell medium in a concentration between 1 ng/ml to 50 ng/ml, preferably between 5 ng/ml and 15 ng/ml (such as 5 ng/ml, 6 ng/ml, 7 ng/ml, 8 ng/ml, 9 ng/ml, 10 ng/ml, 11 ng/ml, 12 ng/ml, 13 ng/ml, 14 ng/ml or 15 ng/ml).

"Primed MSCs" in the present invention refers to MSCs that have undergone "priming". "TNF-o primed MSCs" for instance, refers to MSCs that have been primed with TNF-o. As discussed above, MSCs primed with inflammatory cytokines or mediators have a different expression and/or secretion of anti-inflammatory and immunomodulatory factors. "Renal primed MSCs" in the present invention refers to MSCs that have been primed in order to enhance their renal-protective properties. Such renal primed MSCs can for instance secrete many different cytokines and growth factors, which regulate immune activity and enhance the potential of expansion and differentiation of host cells, thus promoting the recovery of damaged renal tissues. Such renal primed MSCs can for instance improve hematuria, elevated blood pressure, decreased urine output, and/or edema in subjects suffering from kidney disease. As such, renal primed MSCs can be used for the treatment of kidney disease (for instance chronic kidney disease) in an affected subject.

"Mixed Lymphocyte Reaction (MLR)" assays are traditionally used to investigate if an external agent stimulates or suppresses T-cell proliferation. By using a MLR assay, the immunomodulatory properties of the MSCs can be investigated. For this MLR assay the responder T-cells are marked with a fluorescent dye which lights up green when it is exposed to a specific light frequency. These responder T-cells are then stimulated with a plant mitogen Concanavalin A (ConA) to induce or stimulate proliferation. ConA is an antigen-independent mitogen and can be used as an alternative T cell stimulus. This lectin is frequently used as a surrogate for antigen- presenting cells in T cell stimulation experiments. Concanavalin A irreversibly binds to glycoproteins on the cell surface and commits T cells to proliferation. This is a quick way to stimulate transcription factors and cytokine production. When the T- cells start to divide the dye is distributed over their daughter cells, so the dye is serially diluting with every cell division. Therefore, the amount of proliferation of T- cells can be measured by looking at the decrease of colour. Thus, to investigate the immunomodulatory properties of the MSCs, these MSCs are added to the stimulated responder T-cells and co-incubated for several days. Appropriate positive and negative controls are included to see if the test is performed successfully. At the end of the incubation period, the amount of T-cell proliferation is measured using flow cytometry, enabling to see whether or not the MSCs suppressed the T-cell proliferation.

Description

Chronic kidney disease (CKD) is the persistent loss of kidney function over time. It is one of the most common conditions affecting older cats, although it can be seen in animals of any age. Animals with CKD may experience a buildup of waste products and other compounds in the bloodstream that are normally removed or regulated by the kidneys. This accumulation may make them feel ill and appear lethargic, unkempt, and lose weight. They may also lose the ability to concentrate their urine appropriately, and as a result they may urinate greater volumes and drink more water to compensate. The loss of important proteins and vitamins in their urine may contribute to abnormal metabolism and loss of appetite. They may also experience elevated blood pressure (hypertension), which can affect the function of a number of important systems, including the eyes, brain, and heart. Another cause of lethargy in cats with CKD is the buildup of acids in their blood. Their affected kidneys may not excrete these compounds appropriately, making the animals prone to blood acidification, or acidosis, a condition that can significantly affect the function of a variety of organ systems in the body. CKD may also decrease an animal's ability to produce red blood cells, which can lead to anemia, a reduced concentration of red blood cells in their blood. This may cause their gums to appear pale pink, or in severe cases, whitish in color, and may make them lethargic.

At the moment, there is no definitive cure for CKD, however some treatments can improve and prolong the lives of these animals suffering from CKD. Therapy is generally geared toward minimizing the buildup of toxic waste products in the bloodstream, maintaining adequate hydration, addressing disturbances in electrolyte concentration, supporting appropriate nutrition, controlling blood pressure, and slowing the progression of kidney disease.

Dietary modification is an important and proven aspect of CKD treatment. However, many cats have difficulty accepting therapeutic diets, so owners must be patient and dedicated to sticking to the plan. Anemia in a cat with CKD may be treated by replacement therapy with erythropoietin (or with related compounds), which stimulates red blood cell production. In some cases, blood transfusions, which may be used to restore normal red blood cell concentrations using blood obtained from a donor animal, may be necessary.

To date, no known treatments that stop disease progression or repair affected kidneys have been identified.

MSCs have been investigated in cell-based therapies because of their remarkable anti-inflammatory, immunosuppressive, immunomodulatory, and regenerative properties, which involve both paracrine and cell-to-cell contact mechanisms. Paracrine effects depend on the MSC secretome, which includes many bioactive molecules, such as growth factors, cytokines, chemokines, and microvesicles/exosomes carrying proteins and/or miRNAs to target cells. The MSC secretome also contains large amounts of immunoregulatory factors, which are capable of modulating innate and adaptive immune responses.

Mesenchymal stem cells (MSC) are also being explored as a treatment for CKD in both people and animals. Mesenchymal stem cells (MSCs) are isolated from diverse tissues, including bone marrow and adipose tissue. They have the characteristics of multipotent cells, with multi-lineage differentiation, self-renewal, and proliferative potential. MSCs can secrete many different cytokines and growth factors, which regulate immune activity and enhance the potential of expansion and differentiation of host cells, thus promoting the recovery of damaged tissues. They also play critical roles in the modulation of renal blood flow, capillary permeability, endothelial cell survival, and immunological responses. Therefore, MSCs with potential angiogenic and immunomodulatory properties, are also a promising source of cells for the recovery of damaged sites and the treatment of various pathological conditions, such as renal injury and renal failure, making them an ideal therapeutic strategy for regenerative kidney therapy.

Several feline studies have investigated their safety and efficacy in treatment and showed interesting results. A study in eight CKD cats however suggested that allogeneic MSCs from cryopreserved adipose tissue had no improvement after administration (Quimby, Jessica M et al. "Assessment of intravenous adipose- derived allogeneic mesenchymal stem cells for the treatment of feline chronic kidney disease: a randomized, placebo-controlled clinical trial in eight cats." Journal of feline medicine and surgery vol. 18,2 (2016): 165-71. doi: 10.1177/1098612X15576980).

To date, several studies have demonstrated that the modulation of biological, biochemical, and/or biophysical factors can influence MSC fate, lineage-specific differentiation, and functions and also enhance their therapeutic potential. One of the first reported approaches was cell priming (also referred to as licensing or preconditioning) with pro-inflammatory mediators. Cell priming consists of preparing cells for some specific function or lineage-specific differentiation, which involves cell activation, molecular signaling, genetic or epigenetic modifications, and morphology/phenotype changes. This concept is commonly used in the immunology field, and it has been adapted for the stem cell scope. For example, a pro- inflammatory cytokine may be added to the medium during MSC culture to augment their anti-inflammatory effects. Often interferon-y (IFN-y) is used to prime the MSCs. However, several studies have demonstrated that priming with IFN-y leads to upregulation of class I and class II HLA molecules, which makes them more immunogenic and therefore more susceptible to recognition by host immune cells, and subsequently, there is rapid clearance in vivo following administration, especially in xenogeneic transplantation settings.

As such, priming of MSCs is known from the prior art, however in vivo studies using primed MSCs are limited. Importantly, the use of primed MSCs for the treatment of CKD (in cats) has not previously been investigated. The inventors surprisingly found that priming by in vitro culturing said MSCs in an inducing cell medium comprising TNF-o enhances the therapeutic potential of the MSCs.

In a first aspect, the present invention relates to primed mesenchymal stem cells (MSCs) or a pharmaceutical composition comprising a therapeutically effective amount of said primed MSCs for use in the treatment of chronic kidney disease in an affected feline, wherein said composition is administered to an affected feline and wherein prior to said administration the MSCs are primed by in vitro culturing said MSCs in an inducing cell medium comprising TNF-o. Said feline may be any cat of the Felidae family, preferably of the Felinae subfamily, more preferably a domestic cat (Felis catus).

In an embodiment, TNF-o is present in said inducing cell medium in a concentration between 1 ng/ml to 50 ng/ml, preferably between 5 ng/ml and 15 ng/ml, such as 10 ng/ml. TNF-o could be for instance present in said inducing cell medium in a concentration of 1 ng/ml, 2 ng/ml, 3 ng/ml, 4 ng/ml, 5 ng/ml, 6 ng/ml, 7 ng/ml, 8 ng/ml, 9 ng/ml, 10 ng/ml, 11 ng/ml, 12 ng/ml, 13 ng/ml, 14 ng/ml, 15 ng/ml, 16 ng/ml, 17 ng/ml, 18 ng/ml, 19 ng/ml, 20 ng/ml, 21 ng/ml, 22 ng/ml, 23 ng/ml, 24 ng/ml, 25 ng/ml, 26 ng/ml, 27 ng/ml, 28 ng/ml, 29 ng/ml, 30 ng/ml, 31 ng/ml, 32 ng/ml, 33 ng/ml, 34 ng/ml, 35 ng/ml, 36 ng/ml, 37 ng/ml, 38 ng/ml, 39 ng/ml, 40 ng, 41 ng/ml, 42 ng/ml, 43 ng/ml, 44 ng/ml, 45 ng/ml, 46 ng/ml, 47 ng/ml, 48 ng/ml, 49 ng/ml or 50 ng/ml.

By priming said MSCs with TNF-o prior to administration, the immunomodulatory properties of the MSCs are enhanced. Such primed MSCs can for instance secrete many different cytokines and growth factors, which regulate immune activity and enhance the potential of expansion and differentiation of host cells, thus promoting the recovery of damaged renal tissues and enhancing the therapeutic potential of the MSCs.

The combination of various inflammatory cytokines for priming MSCs may lead to additional effects. As such, in an embodiment, said inducing cell medium further comprises interleukin (IL)-ip. In an embodiment, IL- ip is present in said inducing cell medium in a concentration between 1 ng/ml to 50 ng/ml, preferably between 5 ng/ml and 15 ng/ml, such as 10 ng/ml. IL- 13 could be for instance present in said inducing cell medium in a concentration of 1 ng/ml, 2 ng/ml, 3 ng/ml, 4 ng/ml, 5 ng/ml, 6 ng/ml, 7 ng/ml, 8 ng/ml, 9 ng/ml, 10 ng/ml, 11 ng/ml, 12 ng/ml, 13 ng/ml, 14 ng/ml, 15 ng/ml, 16 ng/ml, 17 ng/ml, 18 ng/ml, 19 ng/ml, 20 ng/ml, 21 ng/ml,

22 ng/ml, 23 ng/ml, 24 ng/ml, 25 ng/ml, 26 ng/ml, 27 ng/ml, 28 ng/ml, 29 ng/ml,

30 ng/ml, 31 ng/ml, 32 ng/ml, 33 ng/ml, 34 ng/ml, 35 ng/ml, 36 ng/ml, 37 ng/ml,

38 ng/ml, 39 ng/ml, 40 ng, 41 ng/ml, 42 ng/ml, 43 ng/ml, 44 ng/ml, 45 ng/ml, 46 ng/ml, 47 ng/ml, 48 ng/ml, 49 ng/ml or 50 ng/ml.

In an embodiment, TNF-o is present in said inducing cell medium in a concentration of 1 ng/ml, 2 ng/ml, 3 ng/ml, 4 ng/ml, 5 ng/ml, 6 ng/ml, 7 ng/ml, 8 ng/ml, 9 ng/ml, 10 ng/ml, 11 ng/ml, 12 ng/ml, 13 ng/ml, 14 ng/ml, 15 ng/ml, 16 ng/ml, 17 ng/ml,

18 ng/ml, 19 ng/ml, 20 ng/ml, 21 ng/ml, 22 ng/ml, 23 ng/ml, 24 ng/ml, 25 ng/ml,

26 ng/ml, 27 ng/ml, 28 ng/ml, 29 ng/ml, 30 ng/ml, 31 ng/ml, 32 ng/ml, 33 ng/ml,

34 ng/ml, 35 ng/ml, 36 ng/ml, 37 ng/ml, 38 ng/ml, 39 ng/ml, 40 ng, 41 ng/ml, 42 ng/ml, 43 ng/ml, 44 ng/ml, 45 ng/ml, 46 ng/ml, 47 ng/ml, 48 ng/ml, 49 ng/ml or 50 ng/ml and IL-13 is present in said inducing cell medium in a concentration of 1 ng/ml, 2 ng/ml, 3 ng/ml, 4 ng/ml, 5 ng/ml, 6 ng/ml, 7 ng/ml, 8 ng/ml, 9 ng/ml, 10 ng/ml, 11 ng/ml, 12 ng/ml, 13 ng/ml, 14 ng/ml, 15 ng/ml, 16 ng/ml, 17 ng/ml, 18 ng/ml, 19 ng/ml, 20 ng/ml, 21 ng/ml, 22 ng/ml, 23 ng/ml, 24 ng/ml, 25 ng/ml, 26 ng/ml, 27 ng/ml, 28 ng/ml, 29 ng/ml, 30 ng/ml, 31 ng/ml, 32 ng/ml, 33 ng/ml, 34 ng/ml, 35 ng/ml, 36 ng/ml, 37 ng/ml, 38 ng/ml, 39 ng/ml, 40 ng, 41 ng/ml, 42 ng/ml, 43 ng/ml, 44 ng/ml, 45 ng/ml, 46 ng/ml, 47 ng/ml, 48 ng/ml, 49 ng/ml or 50 ng/ml.

In an embodiment, TNF-o and IL-ip are present in said inducing cell medium in a different concentration. In an embodiment, the TNF-o concentration is higher in the inducing cell medium than the IL-13 concentration. In an embodiment, the TNF-o concentration is lower in the inducing cell medium than the IL-13 concentration. In a preferred embodiment both TNF-o and IL-13 are present in said inducing cell medium in a concentration between 5 ng/ml and 15 ng/ml. In an embodiment, TNF- a is present in said inducing cell medium in a concentration of 5 ng/ml and IL-10 is present in said inducing cell medium in a concentration of 5 ng/ml, 6 ng/ml, 7 ng/ml,

8 ng/ml, 9 ng/ml, 10 ng/ml, 11 ng/ml, 12 ng/ml, 13 ng/ml, 14 ng/ml or 15 ng/ml. In an embodiment, TNF-o is present in said inducing cell medium in a concentration of 6 ng/ml and IL- 13 is present in said inducing cell medium in a concentration of 5 ng/ml, 6 ng/ml, 7 ng/ml, 8 ng/ml, 9 ng/ml, 10 ng/ml, 11 ng/ml, 12 ng/ml, 13 ng/ml, 14 ng/ml or 15 ng/ml. In an embodiment, TNF-o is present in said inducing cell medium in a concentration of 7 ng/ml and IL-13 is present in said inducing cell medium in a concentration of 5 ng/ml, 6 ng/ml, 7 ng/ml, 8 ng/ml, 9 ng/ml, 10 ng/ml, 11 ng/ml, 12 ng/ml, 13 ng/ml, 14 ng/ml or 15 ng/ml. In an embodiment, TNF-o is present in said inducing cell medium in a concentration of 8 ng/ml and IL- 10 is present in said inducing cell medium in a concentration of 5 ng/ml, 6 ng/ml, 7 ng/ml, 8 ng/ml, 9 ng/ml, 10 ng/ml, 11 ng/ml, 12 ng/ml, 13 ng/ml, 14 ng/ml or 15 ng/ml. In an embodiment, TNF-o is present in said inducing cell medium in a concentration of 9 ng/ml and IL-10 is present in said inducing cell medium in a concentration of 5 ng/ml, 6 ng/ml, 7 ng/ml, 8 ng/ml, 9 ng/ml, 10 ng/ml, 11 ng/ml, 12 ng/ml, 13 ng/ml, 14 ng/ml or 15 ng/ml. In an embodiment, TNF-o is present in said inducing cell medium in a concentration of 10 ng/ml and IL-10 is present in said inducing cell medium in a concentration of 5 ng/ml, 6 ng/ml, 7 ng/ml, 8 ng/ml,

9 ng/ml, 10 ng/ml, 11 ng/ml, 12 ng/ml, 13 ng/ml, 14 ng/ml or 15 ng/ml. In an embodiment, TNF-o is present in said inducing cell medium in a concentration of 11 ng/ml and IL-10 is present in said inducing cell medium in a concentration of 5 ng/ml, 6 ng/ml, 7 ng/ml, 8 ng/ml, 9 ng/ml, 10 ng/ml, 11 ng/ml, 12 ng/ml, 13 ng/ml, 14 ng/ml or 15 ng/ml. In an embodiment, TNF-o is present in said inducing cell medium in a concentration of 12 ng/ml and IL-10 is present in said inducing cell medium in a concentration of 5 ng/ml, 6 ng/ml, 7 ng/ml, 8 ng/ml, 9 ng/ml, 10 ng/ml, 11 ng/ml, 12 ng/ml, 13 ng/ml, 14 ng/ml or 15 ng/ml. In an embodiment, TNF-o is present in said inducing cell medium in a concentration of 13 ng/ml and IL-

10 is present in said inducing cell medium in a concentration of 5 ng/ml, 6 ng/ml, 7 ng/ml, 8 ng/ml, 9 ng/ml, 10 ng/ml, 11 ng/ml, 12 ng/ml, 13 ng/ml, 14 ng/ml or 15 ng/ml. In an embodiment, TNF-o is present in said inducing cell medium in a concentration of 14 ng/ml and IL-10 is present in said inducing cell medium in a concentration of 5 ng/ml, 6 ng/ml, 7 ng/ml, 8 ng/ml, 9 ng/ml, 10 ng/ml, 11 ng/ml, 12 ng/ml, 13 ng/ml, 14 ng/ml or 15 ng/ml. In an embodiment, TNF-o is present in said inducing cell medium in a concentration of 15 ng/ml and IL-10 is present in said inducing cell medium in a concentration of 5 ng/ml, 6 ng/ml, 7 ng/ml, 8 ng/ml, 9 ng/ml, 10 ng/ml, 11 ng/ml, 12 ng/ml, 13 ng/ml, 14 ng/ml or 15 ng/ml. TNF-o and IL- ip could for instance both be present in said inducing cell medium in a concentration of 5 ng/ml, 6 ng/ml, 7 ng/ml, 8 ng/ml, 9 ng/ml, 10 ng/ml, 11 ng/ml, 12 ng/ml, 13 ng/ml, 14 ng/ml or 15 ng/ml. In a preferred embodiment, TNF-o and IL- 13 are both present in said inducing cell medium in a concentration of 10 ng/ml.

As described above, by in vitro culturing said MSCs in an inducing cell medium as described in the current invention, comprising TNF-o (and IL-ip), the immunomodulatory properties of the MSCs are increased, for instance the immunosuppressive function of the MSCs is improved and their expression and/or secretion of anti-inflammatory and immunomodulatory factors are increased.

In a further aspect, the invention relates to a method for increasing the immunomodulatory properties of MSCs, said method comprises in vitro culturing said MSCs in an inducing cell medium comprising TNF-o (and as such priming said MSCs). In a preferred embodiment, TNF-o is present in said inducing cell medium in a concentration between 1 ng/ml to 50 ng/ml, preferably between 5 ng/ml and 15 ng/ml. In a preferred embodiment, said cultured MSCs are derived from blood, preferably peripheral blood. In an embodiment, IL-ip is added to the inducing cell medium. In a preferred embodiment, IL-ip is present in said inducing cell medium in a concentration between 1 ng/ml to 50 ng/ml, preferably between 5 ng/ml and 15 ng/ml.

For instance, said primed MSCs might have an increased expression and/or secretion of the immunomodulatory prostaglandin E₂ cytokine, contributing to their immunosuppressive function. In an embodiment, said primed MSCs have an increased secretion of the immunomodulatory prostaglandin E₂ cytokine. In a preferred embodiment, said primed MSCs have an increased secretion of the immunomodulatory prostaglandin E₂ cytokine compared to unprimed MSCs. In a preferred embodiment, said increased secretion comprises a fold change of at least 2, preferably at least 3, preferably at least 4, preferably at least 5, preferably at least 6, preferably at least 7, preferably at least 8, preferably at least 9.

The inventors found that preconditioning or priming with TNF-o is capable to increase secretion of the immunomodulatory prostaglandin E₂ cytokine even more compared to the amount of PGE₂ secretion by unprimed MSCs and that preconditioning or priming with both TNF-o and IL-ip is capable to increase secretion of the immunomodulatory prostaglandin E₂ cytokine even more compared to the PGE₂ secretion by unprimed MSCs and compared to the PGE₂ secretion by MSCs primed with solely TNF-a. Likewise, secretion of the anti-inflammatory cytokine IL-6 is enhanced compared to secretion by unprimed MSCs.

In an embodiment, said primed MSCs have already an increased secretion of PGE₂ when present in basal conditions, so without any stimulation, compared to unprimed MSCs. In an embodiment, said primed MSCs have an increased secretion of PGE₂ compared to unprimed MSCs, when present in an inflammatory environment or condition.

Inflammatory environments or conditions are characterized by the recruitment of immune cells of the blood. Inflammatory mediators include prostaglandins, inflammatory cytokines such as IL-ip, TNF-o, IL-6 and IL-15, chemokines such as IL-8 and other inflammatory proteins like TNF-o, IFN-y. These mediators are primarily produced by monocytes, macrophages, T-cells, B-cells to recruit leukocytes at the site of inflammation and subsequently stimulate a complex network of stimulatory and inhibitory interactions to simultaneously destruct and heal the tissue from the inflammatory process.

Prostaglandin E₂ (PGE₂) is a subtype of the prostaglandin family. PGE₂ is synthesized from arachidonic acid (AA) released from membrane phospholipids through sequential enzymatic reactions. Cyclooxygenase-2 (COX-2), known as prostaglandin-endoperoxidase synthase, converts AA to prostaglandin H₂ (PGH₂), and PGE₂ synthase isomerizes PGH₂ to PGE₂. As a rate-limiting enzyme, COX-2 controls PGE₂ synthesis in response to physiological conditions, including stimulation by growth factors, inflammatory cytokines and tumor promoters.

In a particular embodiment, said primed MSCs present in an inflammatory environment secrete the soluble immune factor prostaglandin E₂ (PGE₂) in a concentration ranging between 1 x 10³ to 1 x 10⁷ picogram per ml, preferably between 1 x 10³ and 5 x 10⁶ picogram per ml, preferably between 1 x 10³ and 2 x 10⁶ picogram per ml to induce or stimulate MSC-regulated immunosuppression.

The PGE₂ secretion of the primed MSCs in those specific concentration ranges stimulates anti-inflammatory processes in vitro and in vivo.

In an embodiment, the primed MSCs for use according to the current invention show an increased expression and/or secretion of various other anti-inflammatory factors, such as anti-inflammatory cytokines. In another or further embodiment, the primed MSCs for use according to the invention, have an increased secretion of at least one of the molecules chosen from IL-6, IL-10, TGF-beta, NO or a combination thereof, and a decreased secretion of IL-lo compared to unprimed MSCs. In an embodiment, the primed MSCs for use according to the current invention show an increased expression and/or secretion of the anti-inflammatory cytokine IL-6. In an embodiment, said expression is measured on the the RNA level, for instance by RNA sequencing. In an embodiment, said expression and/or secretion is measured on the protein level, for instance by ELISA.

In a preferred embodiment, the MSCs have an increased secretion of at least one of the molecules chosen of IL-6, IL-10, TGF-p, NO, or a combination thereof, and a decreased secretion of IL-lo. Comparison can be made with a mesenchymal stem cell having the same characteristics as presented above, but which is not primed.

Preferably the MSCs have an increased secretion of PGE₂ in combination with two or more of the abovementioned factors.

PGE₂, IL-6, IL-10, TGF-p and NO help suppress the proliferation and function of major immune cell populations like T cells and B cells. In addition, the MSCs express low levels of MHC class I molecules and/or are negative for MHC class II molecules on their surface, escaping immunogenic reactions. In addition, the MSCs can suppress the proliferation of white blood cells by their increased secretion of abovementioned factors, once again helping to avoid immunogenic reactions of the host. "Measure negative for MHC class II molecules" refers to an expression of said marker below 2% (the acceptance range is between 0-2%).

In another or further embodiment the primed MSCs stimulate the secretion of PGE₂, IL-6, IL-10, NO, or a combination thereof and/or suppress the secretion of TNF-o, IFN-y, IL-1, IL-13, or a combination thereof in the presence of peripheral blood mononuclear cells (PBMCs). In another or further embodiment, the primed MSCs suppress the secretion of TGF-pi in the presence of PBMCs.

The primed MSCs secrete multiple factors that modulate the immune response of the host. In addition, the primed MSCs have the stimulatory effect to induce or stimulate the secretion of one or more factors selected from the group consisting of PGE₂, IL-6, IL-10, NO, or a combination thereof. Next to the immunomodulatory effect of the primed MSCs on the PBMCs, the primed MSCs also have a suppressive effect on the secretion of the PBMCs, resulting in a decrease of one or more factors selected from the group consisting of TNF-o, IFN-y, IL-1, TGF-pi, IL-13, or a combination thereof. The primed MSCs have a regulatory effect in the inflammatory environment, making them useful in the treatment of all sorts of diseases, particularly disorders of the immune system.

Immunomodulatory properties of the primed MSCs may be assayed using an MLR assay. For this MLR assay responder T-cells are marked with a fluorescent dye which lights up green when it is exposed to a specific light frequency. These responder T- cells are then stimulated with a plant mitogen (ConA) to induce or stimulate proliferation. When the T-cells start to divide the dye is distributed over their daughter cells, so the dye is serially diluting with every cell division. Therefore, the amount of proliferation of T-cells can be measured by looking at the decrease of color. Thus, to investigate the immunomodulatory properties of the primed MSCs, these primed MSCs are added to the stimulated responder T-cells and co-incubated for several days. Appropriate positive and negative controls are included to see if the test is performed successfully. At the end of the incubation period, the amount of T-cell proliferation is measured using flow cytometry, enabling us to see whether or not the MSCs suppressed the T-cell proliferation.

Relevant biological features of the primed MSCs (such as gene expression, protein expression and protein secretion) can be identified by using technologies such as flow cytometry, immunocytochemistry, mass spectrometry, gel electrophoresis, an immunoassay (e.g. immunoblot, Western blot, immunoprecipitation, ELISA), nucleic acid amplification (e.g. real time RT-PCR), enzymatic activity, omics-technologies (proteomics, lipidomics, glycomics, translatomics, transcriptomics, metabolomics) and/or other biological activity.

Furthermore, unlike the use of MSCs which are primed by in vitro culturing said MSCs in other inducing cell media (for instance comprising IFN-y), the current invention uses MSCs which show a low level of immunogenicity, making them less susceptible to recognition by host immune cells. Nor are pro-inflammatory pathways upregulated by the primed MSCs, which is often the case when the MSCs would be primed by other pro-inflammatory cytokines.

In an embodiment, the primed MSCs for use according to the current invention show a decreased activation of pro-inflammatory pathways compared to alternatively primed MSCs. This is important, given that the administered MSCs for use according to the current invention should not elicit inflammation in the treatment of chronic kidney disease.

In an embodiment, the MSCs for use of the present invention may be characterized by the presence of/are measured positive for one or more of the following markers CD29, CD44, CD90, CD105, vimentin, fibronectin, Ki67, CK18 or any combination thereof. In a further embodiment, the MSCs for use of current invention may be characterized by the presence of mesenchymal markers CD29, CD44 and CD90. By means of the latter, the purity of the obtained MSCs can be analyzed, and the percentage of MSCs can be determined.

In general, any technology for identifying and characterizing cellular markers for a specific cell type (e.g. mesenchymal, hepatic, hematopoietic, epithelial, endothelial markers) or having a specific localization (e.g. intracellular, on cell surface, or secreted) that are published in the literature may be considered appropriate for characterizing MSCs. Such technologies may be grouped in two categories: those that allow maintaining cell integrity during the analysis, and those based on extracts (comprising proteins, nucleic acids, membranes, etc.) that are generated using such cells. Among the technologies for identifying such markers and measuring them as being positive or negative, immunocytochemistry or analysis of cell culture media are preferred since these allow marker detection even with the low amount of cells, without destroying them (as it would be in the case of Western Blot or Flow Cytometry).

CD29 is a cell surface receptor encoded by the integrin beta 1 gene, wherein the receptor forms complexes with other proteins to regulating physiological activities upon binding of ligands. The CD44 antigen is a cell surface glycoprotein involved in cell-cell interactions, cell adhesion and migration. In addition, is CD44 a receptor for hyaluronic acid and can also interact with other ligands such as osteopontin, collagens and matrix metalloproteinases (MMPs). The CD90 antigen is a conserved cell surface protein considered as a marker for stem cells, like MSCs. The MSCs of current invention being triple positive for CD29/CD44/CD90 enables the person skilled in the art for a fast and unambiguous selection of the MSCs and provides the MSCs biological properties which are of interest for further downstream applications.

In an embodiment, the MSCs for use of the current invention are characterized by the absence of/measure negative for Major Histocompatibility Complex (MHC) class II molecules, preferably all currently known MHC Class II molecules, classifying the cell as a cell that can be used in cellular therapy for mammalians, such as feline cellular therapy. Even when the MSCs are partly differentiated, the MSCs remain negative for MHC class II molecules. Detecting presence or absence, and quantifying the expression of MHC II molecules can be performed using flow cytometry.

In another and further embodiment the MSCs measure negative for CD45 antigen, a marker for hematopoietic cells.

In an embodiment, the MSCs measure negative for both MHC class II molecules and CD45. "Measure negative for MHC class II molecules" refers to an expression of said marker below 2% (the acceptance range is between 0-2%). "Measure negative for CD45" refers to an expression of said marker below 5% (the acceptance range is between 0-5%).

In a particularly preferred embodiment, the MSCs for use of the current invention measure positive for mesenchymal markers CD29, CD44 and CD90 and measure negative for MHC class II molecules and CD45.

MSCs in general express MHC Class I antigen on their surface. In a most preferred embodiment said MSCs measure negative for MHC Class II markers and have a lower level of MHC Class I marker, wherein said cell exhibits an extremely low immunogenic phenotype. Detecting presence or absence, and quantifying the expression of MHC I and MHC II molecules can be performed using flow cytometry.

These immunological properties of the MSCs limit the ability of the recipient immune system to recognize and reject cells, preferably allogeneic or xenogeneic cells, following cellular transplantation. The production of factors by MSCs, that modulate the immune response together with their ability to differentiate into appropriate cell types under local stimuli make them desirable stem cells for cellular therapy.

In a preferred embodiment the MSCs for use of the current invention measures: positive for mesenchymal markers CD29, CD44 and CD90; positive for one or more markers comprised in the group consisting of vimentin, fibronectin, Ki67, or a combination thereof; negative for MHC class II molecules; negative for hematopoietic marker CD45, and preferably have a low or undetectable level of MHC Class I molecules In a most preferred embodiment, the MSCs for use of the current invention measures: positive for mesenchymal markers CD29, CD44 and CD90; positive for one or more markers comprised in the group consisting of vimentin, fibronectin, Ki67, or a combination thereof; negative for MHC class II molecules; negative for hematopoietic marker CD45; and preferably have a low or undetectable level of MHC Class I molecules, wherein said cell secretes immunomodulatory PGE₂ cytokine in a concentration ranging between 1 x 10³ to 2 x 10⁶ picogram per ml when present in an inflammatory environment or condition.

By preference, the MSCs have a cell size between 10 pm to 100 pm, more preferably between 15 pm and 80 pm, more preferably between 20 pm and 75 pm, more preferably between 25 pm and 50 pm. In an embodiment, the MSCs for use according to the current invention are selected by size by means of a filter system, wherein the cells are run through a double filtration step using a 40 pm filter. Double or multiple filtration steps are preferred. The latter provides for a high population of single cells and avoids the presence of cell aggregates. Such cell aggregates may cause cell death during the preservation of the cells by freezing and may all have an impact on further downstream applications of the cells. For instance, cell aggregates may higher the risk of the occurrence of a capillary embolism when administered intravenously.

The MSCs for use according to the present invention may originate from various tissues or body fluids, in particular from blood, bone marrow, fat tissue or amniotic tissue. Bone marrow harvesting of MSCs has been reportedly associated with haemorrhage, chronic pain, neurovascular injury, and even death. Adipose tissue as a source for MSCs is regarded as a safer option. However, harvesting of MSCs from adipose tissue still requires an incision in the donor animal, hence this is still an invasive procedure. MSCs derived from blood show similar morphology as MSCs derived from bone marrow and adipose tissue. As a consequence, by preference, the MSCs originate from blood, including but not limited to umbilical cord blood and peripheral blood. More preferably, the MSCs originate from peripheral blood. Blood is not only a non-invasive and painless source, but also simple and safe to collect and, consequently, easily accessible. The MSCs or blood comprising MSCs may originate from all mammals, including, but not limited to, humans, domestic and farm animals, zoo animals, sport animals, pet animals, companion animals and experimental animals, such as, for example, mice, rats, rabbits, dogs, cats, cows, horses, pigs and primates, e.g., monkeys and apes; especially horse, human, cat, dogs, rodents, etc. In an embodiment, said origin is equine. In particular, MSCs may be derived from peripheral blood, preferably equine peripheral blood, which allows multiple MSC collections per year with minimal discomfort or morbidities for the donor animal.

In some cases, the use of allogeneic or xenogeneic MSCs is a more favorable option as they offer a stringent selection of healthy and high-quality stem cell donors. They allow the production of a ready-to-use product, avoiding the invasive harvesting and time-consuming cultivation of MSCs from each individual patient. Because of the relative low culture capacity of feline MSCs compared to for example equine or human MSCs, the use of xenogeneic (e.g. human or equine) MSCs is preferred above allogeneic feline MSCs, especially for commercial applications, such as for use in the treatment of CKD in felines.

Therefore, in a particular embodiment the MSCs of the current invention may be used for allogeneic or xenogeneic administration to a subject. As already indicated, allogeneic or xenogeneic use allows a better control of the quality of the MSCs, as different donors may be screened, and the optimal donors may be selected. The latter is indispensable in view of preparing functional MSCs. This is in contrast to autologous use of MSCs, as in this case, quality of the cells is more difficult to be ensured. Nonetheless, autologous use may have his benefits as well. In one case, blood MSCs are isolated, for which blood from a donor was used who was later also recipient of the isolated MSCs (autologous use). In another case, blood is used from donors in which the donor is either of the same family, gender or race as the recipient of the MSCs isolated from the blood of donors (allogeneic use) or not (xenogeneic use). In particular, these donors will be tested on common current transmittable diseases or pathologies, in order to avoid the risk of horizontal transmission of these pathologies or diseases through the stem cells. Preferably, the donors/donor animals are kept in quarantine. Preferably, the MSCs of the current invention may be used for xenogeneic administration to a subject. When using donor horses they can be, for example tested for the following pathologies, viruses or parasites: equine infectious anemia (EIA), equine rhinopneumonitis (EHV- 1, EHV-4), equine viral arteritis (EVA), West Nile virus (WNV), African horse Sickness (AHS), dourine (Trypanosoma), equine piroplasmosis, glanders (malleus, glanders), equine influenza, Lyme borreliosis (LB) (Borrelia burgdorferi, Lyme disease).

In a further aspect, the invention relates to TNF-o primed MSCs or a composition comprising said TNF-o primed MSCs, wherein said MSCs are preferably equine- derived and derived from blood, preferably peripheral blood. Said MSCs are primed by in vitro culturing said MSCs in an inducing cell medium comprising TNF-o. As described above, TNF-o can preferably be present in said inducing cell medium in a concentration between 1 ng/ml to 50 ng/ml, preferably between 5 ng/ml and 15 ng/ml. In an embodiment, said inducing cell medium further comprises interleukin IL-ip. As described above, IL-ip can preferably be present in said inducing cell medium in a concentration between 1 ng/ml to 50 ng/ml, preferably between 5 ng/ml and 15 ng/ml. The inventors surprisingly found that such TNF-o (and IL- 13) primed equine peripheral blood MSCs (ePB-MSCs) show favourable immunomodulatory properties, also evidenced by improved clinical parameters (for instance: quality of life, blood pressure and body weight) of the affected felines. Said TNF-o (and I L- 1 ) primed ePB-MSCs support the healing process by the release of cytokines, which act upon the inflammation, causing an anti-inflammatory reaction. By in vitro culturing said MSCs in an inducing cell medium as described in the current invention, comprising TNF-o (and IL-ip), the immunomodulatory properties of the MSCs are increased, for instance the immunosuppressive function of the MSCs is improved and their secretion of anti-inflammatory and immunomodulatory factors are increased.

As described above, said primed MSCs might have an increased expression and/or secretion of the immunomodulatory prostaglandin E₂ cytokine, contributing to their immunosuppressive function. The primed MSCs according to the current invention might also show an increased expression and/or secretion of other antiinflammatory factors, such as anti-inflammatory cytokines. In another or further embodiment, the primed MSCs according to the invention, might have an increased secretion of at least one of the molecules chosen from IL-6, IL-10, TGF-beta, NO or a combination thereof, and a decreased secretion of IL-lo compared to unprimed MSCs.

In another or further embodiment the primed MSCs stimulate the secretion of PGE₂, IL-6, IL-10, NO, or a combination thereof and/or suppress the secretion of TNF-o, IFN-y, IL-lo, IL-13, or a combination thereof in the presence of peripheral blood mononuclear cells (PBMCs). In another or further embodiment, the primed MSCs suppress the secretion of TGF-pi in the presence of PBMCs.

In the inflammatory environment the primed MSCs secrete multiple factors that modulate the immune response of the host. In addition, the primed MSCs have the stimulatory effect to induce or stimulate the secretion of one or more factors selected from the group consisting of PGE₂, IL-6, IL-10, NO, or a combination thereof. Next to the stimulatory effect of the primed MSCs on the PBMCs in an inflammatory environment, the primed MSCs also have a suppressive effect on the secretion of the PBMCs, resulting in a decrease of one or more factors selected from the group consisting of TNF-o, IFN-y, IL-lo, TGF-pi, IL-13, or a combination thereof. The primed MSCs have a regulatory effect in the inflammatory environment, making them useful in the treatment of all sorts of diseases, particularly disorders of the immune system.

In a further aspect, the invention relates to renal primed MSCs or a composition comprising said renal primed MSCs, wherein said MSCs are preferably equine- derived and derived from blood, preferably peripheral blood. Such renal primed MSCs aid in the healing of the renal tissues by their immunomodulatory properties, reducing the inflammation present in the affected tissue. In chronic kidney disease, tubulointerstitial inflammation and injury is associated with infiltrating inflammatory cells, such as macrophages. Once present at interstitial sites, these inflammatory cells interact with resident cells and extracellular matrix to generate a proinflammatory microenvironment that amplifies tissues injury and promotes scarring. By reducing said proinflammatory microenvironment, the transition of fibroblast to myofibroblast could for instance be decreased. In an embodiment, said renal primed MSCs are primed by in vitro culturing said MSCs in an inducing cell medium comprising TNF-o. In an embodiment, said renal primed MSCs are primed by in vitro culturing said MSCs in an inducing cell medium comprising TNF-o and IL- 10.

In a preferred embodiment, said renal primed MSCs or composition comprising said renal primed MSCs is used in the treatment of chronic kidney disease in an affected feline. In a preferred embodiment, TNF-o, and IL- ip if present, are present in said inducing cell medium in a concentration between 1 ng/ml to 50 ng/ml, preferably between 5 ng/ml and 15 ng/ml. In an embodiment, said renal primed MSCs are intravenously administered. In an embodiment, a dose of 1 x 10⁵ - l x 10⁷ MSCs per animal is administered. In an embodiment, a single dose is administered, or multiple doses are administered. In an embodiment, said renal primed MSCs have an increased secretion of the immunomodulatory prostaglandin E₂ cytokine compared to MSCs that have not received renal priming.

The primed MSCs of current invention may be derived by any standard protocol known in the art. In an embodiment, said primed MSCs may be obtained via a method wherein the MSCs are isolated from blood or a blood phase and wherein said cells are cultured and expanded in a basal medium, preferably a low glucose medium. In a preferred embodiment, priming of the MSCs in the inducing cell medium occurs during the last in vitro culturing passage of the MSCs.

Basal medium formulation as known in the art include, but are not limited to Eagle's Minimum Essential Medium (MEM), Dulbecco's Modified Eagle's Medium (DMEM), alpha modified Minimum Essential Medium (alpha-MEM), Basal Medium Essential (BME), Iscove's Modified Dulbecco's Medium (IMDM), BGJb medium, F-12 Nutrient Mixture (Ham), Liebovitz L-15, DMEM/F-12, Essential Modified Eagle's Medium (EMEM), RPMI-1640, Medium 199, Waymouth's 10 MB 752/1 or Williams Medium E, and modifications and/or combinations thereof. Compositions of the above basal media are generally known in the art and it is within the skill of one in the art to modify or modulate concentrations of media and/or media supplements as necessary for the cells cultured. A preferred basal medium formulation may be one of those available commercially such as DMEM, which are reported to sustain in vitro culture of MSCs, and including a mixture of growth factors for their appropriate growth, proliferation, maintenance of desired markers and/or biological activity, or long-term storage.

In a preferred embodiment, the basal medium formulation comprises DMEM supplemented with Fetal Bovine Serum (FBS). The primary use of FBS is as a growth supplement for in vitro cell culture, and it is typically added to basal cell culture media at a concentration of 5-10%. In a preferred embodiment, the basal medium formulation comprises DMEM supplemented with 5 -10% Fetal Bovine Serum (FBS), such as DMEM supplemented with 5%, 6%, 7%, 8%, 9% or 10% FBS.

Such basal media formulations contain ingredients necessary for mammal cell development, which are known per se. By means of illustration and not limitation, these ingredients may include inorganic salts (in particular salts containing Na, K, Mg, Ca, Cl, P and possibly Cu, Fe, Se and Zn), physiological buffers (e.g., HEPES, bicarbonate), nucleotides, nucleosides and/or nucleic acid bases, ribose, deoxyribose, amino acids, vitamins, antioxidants (e.g., glutathione) and sources of carbon (e.g. glucose, pyruvate, e.g., sodium pyruvate, acetate, e.g., sodium acetate), etc. It will also be apparent that many media are available as low-glucose formulations with or without sodium pyruvate.

Method for isolating MSCs from blood or a blood phase and culturing and expanding said cells are known in the art and for instance described in WO2014053418 or W02014053420.

In an embodiment, such method for isolating MSCs from blood or a blood phase and culturing and expanding said cells in a low glucose medium may comprise the following steps: a) the collection of one or more blood samples from donors, in a sample vial, coated with an anti-coagulant; b) centrifuging the blood samples to obtain a 3-phase distribution, consisting of a plasma-phase, buffy coat, and erythrocytes phase; c) collecting the buffy coat and loading it on a density gradient; d) collecting of the blood-inter-phase obtained from the density gradient of step c); e) isolating of MSCs from the blood-inter-phase by centrifugation; f) seeding between 2.5 x 10⁵/cm² and 5 x 10⁵/cm² MSCs in culture and keeping them in a low glucose growth medium supplemented with dexamethasone, antibiotics and serum.

In an embodiment, anticoagulants may be supplemented to the MSCs. Non-limiting examples are EDTA or heparin.

The number of seeding is crucial to ultimately obtain a pure and viable population MSCs at an acceptable concentration, as a too dense seeding will lead to massive cell death during expansion and a non-homogenous population of MSCs and a too dispersed seeding will result in little or no colony formation of MSCs, so that expansion is not or hardly possible, or it will take too much time. In both cases the viability of the cells will be negatively influenced.

In a preferred embodiment of current invention, the MSCs have a high cell viability, wherein at least 90%, more preferably at least 95%, most preferably 100% of said cells are viable. The blood-interphase is the source of blood mononuclear cells (BMCs) comprising monocytes, lymphocytes, and MSCs. By preference, the lymphocytes are washed away at 37°C, while the monocytes die within 2 weeks in the absence of cytokines necessary to keep them alive. In this way, the MSCs are purified. The isolation of the MSCs from the blood-inter-phase is preferably done by means of centrifugation of the blood-inter-phase, after which the cell pellet is washed at least once with a suitable buffer, such as a phosphate buffer.

In a further embodiment the MSCs of current invention are negative for monocytes and macrophages, both within a range between 0% and 7.5%.

In particular, the mesenchymal cells are kept at least 2 weeks in growth medium. Preferably, growth medium with 1% dexamethasone is used, as the specific characteristics of the MSCs are kept in said medium.

Following a minimum period of 2 weeks (14 days), preferably 3 weeks (21 days) MSC colonies will become visible in the culture bottles. In a subsequent step g) at least 6 x 10³ stem cells/cm² are transferred to an expansion medium containing low glucose, serum and antibiotics for the purpose of expanding the MSCs. Preferably, the expansion of the MSCs will occur in minimal five cell passages. In this way sufficient cells can be obtained. Preferably, the cells are split at 70% to 80% confluency. The MSCs can be maintained up to 50 passages in culture. After this the risk of loss in vitality, senescence or mutation formation occurs.

In a further embodiment, the population doubling time (PDT) between each passage during expansion of the MSCs should be between 0.7 and 3 days after trypsinization. Said PDT between each passage during expansion of the MSCs is preferably between 0.7 and 2.5 days after trypsinization.

In a preferred embodiment, the MSCs for use according to the invention have a spindle-shaped morphology. The morphological characterization of the MSCs of current invention classifies the cell as an elongated, fibroblast-like, spindle-shaped cell. This type of cell is distinct form other populations of MSCs with small selfrenewing cells which reveal mostly a triangular or star-like cell shape and populations of MSCs with a large, cuboidal or flattened pattern with a prominent nucleus. The selection of MSCs with this specific morphological characteristic along with the biological markers enables the person skilled in the art to isolate the MSCs of current invention. A morphological analysis of cells can easily be performed by a person skilled in the art using phase contrast microscopy. Besides, the size and granularity of MSCs can be evaluated using forward and side scatter diagram in flow cytometry or other techniques known by a person skilled in the art.

In another or further preferred embodiment, the MSCs have a suspension diameter between 10 pm and 100 pm. The MSCs for use of current invention have been selected based on size/suspension diameter. By preference, the MSCs have a cell size between 10 to 100 pm, more preferably between 15 and 80 pm, more preferably 20 and 75 pm, more preferably between 25 and 50 pm. Preferably, the selection of cells based on cell size occurs by a filtration step. For instance, MSCs with a cell concentration ranging between 10³ to 10⁷ MSCs per ml, wherein said cells are preferably diluted in low glucose DMEM medium, are selected by size by means of a filter system, wherein the cells are run through a double filtration step using a 40 pm filter. Double or multiple filtration steps are preferred. The latter provides for a high population of single cells and avoids the presence of cell aggregates. Such cell aggregates may cause cell death during the preservation of the cells by freezing and may all have an impact on further downstream applications of the cells. For instance, cell aggregates may higher the risk of the occurrence of a capillary embolism when administered intravenously.

After isolation, the stem cells are cultivated for a certain number of passages (for instance until passage (P)5) and characterized on viability, morphology, presence of cell surface markers and population doubling time. Afterwards, the cells are further cultivated until the last passage (for instance until P10), trypsinized and resuspended. In a preferred embodiment, priming of the MSCs in the inducing cell medium occurs during the last in vitro culturing passage of the MSCs. In a preferred embodiment, the MSCs are resuspended in Dulbecco's Modified Eagle Medium (DMEM) low glucose with 10% dimethylsulfoxide (DMSO).

In a further aspect, the invention relates to an inducing cell medium for in vitro inducing renal protective properties in MSCs, whereby said inducing cell medium comprises between 5 ng/ml and 15 ng/ml TNF-o. In an embodiment, said inducing cell medium comprises TNF-o in a concentration of 5 ng/ml, 6 ng/ml, 7 ng/ml, 8 ng/ml, 9 ng/ml, 10 ng/ml, 11 ng/ml, 12 ng/ml, 13 ng/ml, 14 ng/ml or 15 ng/ml.

The combination of various inflammatory cytokines for priming MSCs may lead to additional effects. As such, in an embodiment, said inducing cell medium further comprises interleukin (IL)-ip. In an embodiment, IL- ip is present in said inducing cell medium in a concentration between 5 ng/ml and 15 ng/ml, such as 10 ng/ml. In an embodiment, said medium comprises IL-1 p in a concentration of 5 ng/ml, 6 ng/ml, 7 ng/ml, 8 ng/ml, 9 ng/ml, 10 ng/ml, 11 ng/ml, 12 ng/ml, 13 ng/ml, 14 ng/ml or 15 ng/ml.

As discussed above, both TNF-o and IL-ip can both be present in various concentrations.

In an embodiment, TNF-o and IL-ip are present in said inducing cell medium in a different concentration. In an embodiment, the TNF-o concentration is higher in the inducing cell medium than the IL-ip concentration. In an embodiment, the TNF-o concentration is lower in the inducing cell medium than the IL-1 p concentration. In a preferred embodiment both TNF-o and IL-ip are present in said inducing cell medium in a concentration between 5 ng/ml and 15 ng/ml. TNF-o and IL- ip could for instance both be present in said inducing cell medium in a concentration of 5 ng/ml, 6 ng/ml, 7 ng/ml, 8 ng/ml, 9 ng/ml, 10 ng/ml, 11 ng/ml, 12 ng/ml, 13 ng/ml, 14 ng/ml or 15 ng/ml. In a preferred embodiment, TNF-o and IL-1 p are both present in said inducing cell medium in a concentration of 10 ng/ml.

The pharmaceutical composition comprising primed MSCs for use according to the current invention, will by preference be frozen in order to allow long-time storage of the composition. Preferably the composition will be frozen at low and constant temperature, such as a temperature below -20°C. These conditions allow a save storage of the MSCs, and enable the MSCs to keep their biological and morphological characteristics, as well as their high cell viability during storage and once thawed. In a preferred embodiment, the primed MSCs are stored at -80°C in cryovials until further use.

In a more preferred embodiment, the pharmaceutical composition comprising primed MSCs for use of the invention can be stored for at least 6 months at a maximum temperature of -80°C, optionally in liquid nitrogen. A crucial factor in the freezing of the MSCs is a cryogenic medium, in particular comprising DMSO. DMSO prevents ice crystal formation in the medium during the freezing process, but may be toxic to the cells in high concentrations. In a preferred embodiment, the concentration of DMSO comprises up to 20%, more preferably up to 15%, more preferably the concentration of DMSO in the cryogen comprises 10%. The cryogenic medium further comprises low-glucose medium such as low glucose DMEM (Dulbecco's Modified Eagle Medium).

Afterwards, the pharmaceutical composition for use of the invention are preferably thawed before administration at a temperature around room temperature, preferably at a temperature between 20°C and 37°C, more preferably at a temperature between 25°C and 37°C, and in a time span of maximal 20 minutes, preferably maximal 10 minutes, more preferably maximal 5 minutes.

Furthermore, the composition is preferably administered within 2 minutes after thawing, in order to safeguard the vitality of the MSCs.

In a preferred embodiment, the primed MSCs for use according to the present invention are formulated for administration in a subject by means of intravenous injection or infusion.

Intravenous administration is a non-invasive procedure and does not require sedation. Such invasive procedures and/or sedation, which may involve risks, especially for older patients which already are at higher risk in developing chronic kidney disease. Therefore, the systemic administration of MSCs via an intravenous (IV) injection offers substantial benefits in therapy application.

In an embodiment, said therapeutically effective amount of MSCs is between 1 x 10⁵ - 1 x 10⁷ MSCs in said composition.

In an embodiment, a therapeutically effective amount of MSCs is administered to the feline patient, preferably a dose of 1 x 10⁵ - 1 x 10⁷ MSCs per patient is administered. In an embodiment, a single dose is administered.

The minimum therapeutically effective dose that yields a therapeutic benefit to a subject is at least 10⁵ of the MSCs per administration. Preferably, each administration is by intravenous injection and comprises between 1 x 10⁵ to 5 x 10⁵

MSCs per administration, wherein said MSCs preferably are xenogeneic. In an embodiment, said MSCs are administered at least twice, at least three times, at least four times, at least five times, preferably with intervals.

In another or further embodiment, the treatment further comprises: multiple administrations of the MSCs or the composition comprising MSCs, for example multiple intravenous administrations, doses of 1 x 10⁵ - l x 10⁷ MSCs per feline patient, wherein said multiple doses are administered at various time points, including but not limited to one or more of the following time points 1 day apart, 2 days apart, 3 days apart, 4 days apart, 5 days apart, 6 days apart, 7 days (1 week) apart, 2 weeks apart, 3 weeks apart, 4 weeks apart, 5 weeks apart, 6 weeks apart, 7 weeks apart, 8 weeks apart, 3 months apart, 6 months, 9 months apart, and/or 1 year apart. Preferably each dose is administered at least 2 weeks apart, more preferably at least 3 weeks apart, even more preferably at least 4 weeks apart, and most preferably at least 6 weeks apart.

In an embodiment, said composition comprises said MSCs present in a sterile liquid. A non-limiting example of such sterile liquid is a minimal essential medium (MEM), such as Dulbecco's Modified Eagle Medium (DMEM). Said sterile liquid should be safe for intravenous administration, e.g. via injection or infusion, to a mammalian patient.

As non-limiting examples, said sterile liquid is a minimal essential medium, such as a basal medium. Basal medium formulation as known in the art include, but are not limited to Eagle's Minimum Essential Medium (MEM), Dulbecco's Modified Eagle's Medium (DMEM), alpha modified Minimum Essential Medium (alpha-MEM), Basal Medium Essential (BME), Iscove's Modified Dulbecco's Medium (IMDM), BGJb medium, F-12 Nutrient Mixture (Ham), Liebovitz L-15, DMEM/F-12, Essential Modified Eagle's Medium (EMEM), RPMI-1640, Medium 199, Waymouth's 10 MB 752/1 or Williams Medium E, and modifications and/or combinations thereof. Compositions of the above basal media are generally known in the art and it is within the skill of one in the art to modify or modulate concentrations of media and/or media supplements as necessary for the cells cultured. A preferred basal medium formulation may be one of those available commercially such as DMEM, which are reported to sustain in vitro culture of MSCs, and including a mixture of growth factors for their appropriate growth, proliferation, maintenance of desired markers and/or biological activity, or long-term storage. Such basal media formulations contain ingredients necessary for mammal cell development, which are known per se. By means of illustration and not limitation, these ingredients may include inorganic salts (in particular salts containing Na, K, Mg, Ca, Cl, P and possibly Cu, Fe, Se and Zn), physiological buffers (e.g., HEPES, bicarbonate), nucleotides, nucleosides and/or nucleic acid bases, ribose, deoxyribose, amino acids, vitamins, antioxidants (e.g., glutathione) and sources of carbon (e.g. glucose, pyruvate, e.g., sodium pyruvate, acetate, e.g., sodium acetate), etc. It will also be apparent that many media are available as low-glucose formulations with or without sodium pyruvate.

By preference, said composition comprises at least 75%, more preferably at least 80%, even more preferably at least 85%, most preferably at least 90% of single cells and whereby said single cells have a suspension diameter of between 10 pm and 100 pm, more preferably between 15 pm and 80 pm, more preferably between 20 pm and 75 pm, more preferably between 25 pm and 50 pm. As previously mentioned, the diameter of the cells as well as their single-cell nature is crucial for any downstream application, e.g. intravenous administration, and for the vitality of the cells.

By preference, said composition comprises at least 90% MSCs, more preferably it will comprise at least 95% MSCs, more preferably at least 99%, most preferably 100% MSCs.

The volume and concentration of the composition in the form of a sterile liquid comprising the MSCs is preferably adapted for intravenous injection. In an embodiment, the pharmaceutical composition may be administered to the animal in the form of a sterile liquid comprising, after final adjustment, the MSCs at a concentration of 1 x 10⁵ - l x 10⁷ cells per mL.

In an embodiment, with each intravenous injection or infusion, a therapeutically effective amount of MSCs is administered, preferably each injection or infusion comprises a dose of 1 x 10⁵ to 1 x 10⁷ of said MSCs.

In an embodiment, the pharmaceutical composition comprises a therapeutically effective of amount of MSCs of between 1 x 10⁵ - l x 10⁷ MSCs per mL, preferably 1 x 10⁵ to 1 x 10⁶ MSCs per mL, more preferably 1 x 10⁵ - 5 x 10⁵ MSCs per mL of said composition, most preferably 3 x 10⁵ MSCs per mL of said composition. In an embodiment, one dosage of said composition has a volume of about 0.5 to 5 ml, preferably of about 0.5 to 5 ml, preferably of about 0.5 to 3 ml, preferably of about 0.5 to 2 ml, more preferably of about 0.5 to 1.5 ml, most preferably of about 1 ml. In another or further embodiment, one dosage of said composition has a volume of maximally about 5 ml, preferably maximally about 4 ml, more preferably maximally about 3 ml, more preferably maximally about 2 ml, most preferably said volume is about 1 ml. This amount is suitable for intravenous administration.

Said dosage may be formulated in a vial or in a pre-filled syringe.

In an embodiment, the volume of the composition which is administered per injection to a patient is adapted in accordance with the patient's body weight. In another an embodiment, a fixed dose of 1 x 10⁵ - l x 10⁷ MSCs per patient, preferably 1 x 10⁵ to 1 x 10⁶ MSCs, more preferably 1 x 10⁵ - 5 x 10⁵ MSCs, most preferably 3 x 10⁵ MSCs is administered.

The inventors have further discovered that a particularly effective treatment is achieved by a dosing regimen comprising at least two dosages of the MSCs for use or the pharmaceutical composition for use as described above in any of the embodiments.

Therefore, a further embodiment relates to a pharmaceutical composition for use in the treatment of CKD in felines, wherein:

- the treatment comprises a step of administering, preferably intravenously, a first amount of said composition comprising a total dose of 1 x 10⁵ - l x 10⁷ MSCs per patient, and

- the treatment further comprises a step of administering, preferably intravenously, a second amount of said composition, said second amount comprising a second total dose of 1 x 10⁵ - l x 10⁷ MSCs, wherein said MSCs preferably are xenogeneic, and wherein said second dose is administered 1 day after the first amount, 2 days after the first amount, 3 days after the first amount, 4 days after the first amount, 5 days after the first amount, 6 days after the first amount, 7 days (1 week) after the first amount, 2 weeks after the first amount, 3 weeks after the first amount, 4 weeks after the first amount, 5 weeks after the first amount, 6 weeks after the first amount, 7 weeks after the first amount, 8 weeks after the first amount, 3 months after the first amount, 6 months, 9 months after the first amount, and/or 1 year after the first amount. Preferably each dose is administered at least 2 weeks after the first amount, more preferably at least 3 weeks after the first amount, even more preferably at least 4 weeks after the first amount, and most preferably at least 6 weeks after the first amount.

In an embodiment, said second dose is identical to the first dose. In another embodiment, said second dose is lower than the first dose. In yet another embodiment, said second dose is higher than the first dose.

In an embodiment, a third, fourth and/or even a fifth amount of said composition may be administered, preferably intravenously, to said patient, wherein said third, fourth and/or fifth amount comprises a third, fourth and/or fifth total dose of 1 x 10⁵ - 1 x 10⁷ MSCs, wherein said MSCs preferably are xenogeneic.

In an embodiment, a sixth or more amount of said composition may be administered, preferably intravenously, to said patient, wherein said sixth or more amount comprises a sixth or more total dose of 1 x 10⁵ - l x 10⁷ MSCs, wherein said MSCs preferably are xenogeneic.

In a further aspect, the invention relates to a pharmaceutical composition comprising peripheral blood-derived MSCs, wherein said MSCs are mammal- derived, preferably equine-derived, and present in a sterile liquid at a concentration of between 1 x 10⁵ - l x 10⁷ MSCs per mL of said composition, wherein said composition has a volume of about 0.5 to 5 ml, wherein said MSCs measure positive for mesenchymal markers CD29, CD44 and CD90 and measure negative for MHC class II molecules and CD45, wherein said MSCs have a suspension diameter between 10 pm and 100 pm and wherein said MSCs are primed by means of an in vitro culturing step in an inducing cell medium comprising TNF-o. In an embodiment, said inducing cell medium further comprises interleukin (IL)-ip.

The favourable immunomodulatory properties and renal-protective properties of the MSCs according to the current invention are evidenced by an enhanced therapeutic effect when administering a therapeutically effective amount of said primed MSCs (for instance compared to administration of unprimed MSCs) in felines diagnosed with or suffering from chronic kidney disease. Such an enhanced therapeutic effect results for instance in an increased quality of life, a more pronounced decrease in blood pressure values, positive effects on the body weight of the treated animals, etc. In an embodiment, by administering the primed MSCs or the pharmaceutical composition comprising a therapeutically effective amount of said primed MSCs, to a feline diagnosed with or suffering from CDK, the creatinine levels in said felines are reduced and/or quality of life (QoL) scores are improved and/or blood pressure is normalized and/or the body weight is normalized, compared to a feline which has not been treated with said primed MSCs or composition and/or compared to baseline values measured before administration of said primed MSCs or the pharmaceutical composition comprising a therapeutically effective amount of said primed MSCs.

In an embodiment, by administering the primed MSCs or the pharmaceutical composition comprising a therapeutically effective amount of said primed MSCs, to a feline diagnosed with or suffering from CDK, the quality of life (QoL) of said feline is improved compared to the quality of life of said feline prior to administration of said pharmaceutical composition comprising primed MSCs. Said improvement of quality of life (QoL) of said feline can occur at any time point after administration of said MSCs or pharmaceutical composition comprising a therapeutically effective amount of said primed MSCs. In an embodiment, said improvement is measured 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years after administration of a first, second, third or further dose of said MSCs or pharmaceutical composition comprising a therapeutically effective amount of said primed MSCs.

Said quality of life can be for instance determined by means of a linear analog scale, asking owners to rate their pets' QoL on a scale of 1-10. When 1 being the best quality of life and 10 being the worst quality of life, improving the quality of life thus refers to obtaining a lower score on said scale. When 1 being the worst quality of life and 10 being the best quality of life, improving the quality of life thus refers to obtaining a higher score on said scale.

In an embodiment, the quality of life (QoL) of said feline is measured by a linear analog scale and the value thus obtained is improved with at least 10%, preferably at least 15%, preferably at least 20%, preferably at least 25%, preferably at least 30%, preferably at least 35%, preferably at least 40%, preferably at least 45%, preferably at least 50%, preferably at least 55%, preferably at least 60%, preferably at least 65%, preferably at least 70%, preferably at least 75%, preferably at least 80%, preferably at least 85%, such as 86%, compared to the value of said feline prior to administration of said pharmaceutical composition comprising primed MSCs.

An "improved value" refers to a better quality of life.

Said quality of life can also be calculated by a scoring scheme or a questionnaire.

In an embodiment, the quality of life of felines is calculated by means of the quality of life (QoL) tool discussed in the article of Bijsmans et al. Bijsmans ES, Jepson RE, Syme HM, Elliott J, Niessen SJ. Psychometric Validation of a General Health Quality of Life Tool for Cats Used to Compare Healthy Cats and Cats with Chronic Kidney Disease. J Vet Intern Med. 2016 Jan-Feb;30(l): 183-91. doi: 10.1111/jvim.13656. Epub 2015 Nov 14. PMID: 26567089; PMCID: PMC4913638.'). In brief, the questionnaire is divided into 4 domains: general health (GH), eating (E), behavior (B) and management (M). Each item is scored according to the frequency or severity with which it impacted the cat's life, and an importance rating is included for all questions to capture individual differences. The frequency or severity ratings range from -3 to +3, and the importance ratings range from 0 to +3. At the end of the calculation series, this results in an average-weighted score that provides an overall quantitative measure of the cat's Quality of Life.

In an embodiment, the quality of life (QoL) of said feline is measured by a scoring scheme and the score thus obtained is improved with at least 10%, preferably at least 15%, preferably at least 20%, preferably at least 25%, preferably at least 30%, preferably at least 35%, preferably at least 40%, preferably at least 45%, preferably at least 50%, preferably at least 55%, preferably at least 60%, preferably at least 65%, preferably at least 70%, preferably at least 75%, preferably at least 80%, preferably at least 85%, such as 86%, compared to the quality of life score of said feline prior to administration of said pharmaceutical composition comprising primed MSCs. An "improved score" refers to a better quality of life.

In an embodiment, the quality of life (QoL) of said feline diagnosed with or suffering from CDK is calculated by means of the quality of life (QoL) tool discussed in the article of Bijsmans et al. and the score thus obtained is improved with at least 10%, preferably at least 15%, preferably at least 20%, preferably at least 25%, preferably at least 30%, preferably at least 35%, preferably at least 40%, preferably at least 45%, preferably at least 50%, preferably at least 55%, preferably at least 60%, preferably at least 65%, preferably at least 70%, preferably at least 75%, preferably at least 80%, preferably at least 85%, such as 86%, compared to the quality of life score of said feline prior to administration of said pharmaceutical composition comprising primed MSCs. An "improved score" refers to a better quality of life.

In an embodiment, by administering the primed MSCs or a pharmaceutical composition comprising a therapeutically effective amount of said primed MSCs, to felines diagnosed with or suffering from CDK, the quality of life (QoL) of said felines is improved compared to the quality of life of felines which have not been treated with said composition.

In an embodiment, the quality of life (QoL) of said felines is measured by a linear analog scale and the mean value thus obtained is improved with at least 10%, preferably at least 15%, preferably at least 20%, preferably at least 25%, preferably at least 30%, preferably at least 35%, preferably at least 40%, preferably at least 45%, preferably at least 50%, preferably at least 55%, preferably at least 60%, preferably at least 65%, preferably at least 70%, preferably at least 75%, preferably at least 80%, preferably at least 85%, such as 86%, compared to the mean value of said felines which have not been treated with said composition.

In an embodiment, the quality of life (QoL) of said felines is measured by a scoring scheme and the mean score thus obtained is improved with at least 10%, preferably at least 15%, preferably at least 20%, preferably at least 25%, preferably at least 30%, preferably at least 35%, preferably at least 40%, preferably at least 45%, preferably at least 50%, preferably at least 55%, preferably at least 60%, preferably at least 65%, preferably at least 70%, preferably at least 75%, preferably at least 80%, preferably at least 85%, such as 86%, compared to the mean quality of life score of said felines which have not been treated with said composition.

In an embodiment, the quality of life (QoL) of said felines diagnosed with or suffering from CDK is measured by means of the quality of life (QoL) tool discussed in the article of Bijsmans et al. and the mean score thus obtained is improved with at least 10%, preferably at least 15%, preferably at least 20%, preferably at least 25%, preferably at least 30%, preferably at least 35%, preferably at least 40%, preferably at least 45%, preferably at least 50%, preferably at least 55%, preferably at least 60%, preferably at least 65%, preferably at least 70%, preferably at least 75%, preferably at least 80%, preferably at least 85%, such as 86%, compared to the mean quality of life score of said felines which have not been treated with said composition. In an embodiment, by administering the primed MSCs or a pharmaceutical composition comprising a therapeutically effective amount of said primed MSCs, to felines diagnosed with or suffering from CDK, the blood pressure of said animals is normalized, and this to a higher degree compared to felines which have not been treated with said primed MSCs or composition. Said improvement in/normalization of blood pressure values of said feline can occur at any time point after administration of said primed MSCs or pharmaceutical composition comprising a therapeutically effective amount of said primed MSCs. In an embodiment, said improvement is measured 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years after administration of a first, second, third or further dose of said primed MSCs or pharmaceutical composition comprising a therapeutically effective amount of said primed MSCs. In an embodiment, said improvement is measured compared to felines which have been treated with unprimed/native MSCs. In an embodiment, said improvement is measured compared to felines which have been treated with a placebo solution. In an embodiment, said blood pressure values are decreased with at least 10 mmHg, preferably at least 20 mmHg, preferably at least 30 mmHg, preferably at least 40 mmHg, preferably at least 50 mmHg, compared to baseline values measured before administration of said primed MSCs or pharmaceutical composition comprising a therapeutically effective amount of said primed MSCs.

In an embodiment, by administering the primed MSCs or a pharmaceutical composition comprising a therapeutically effective amount of said primed MSCs, to felines diagnosed with or suffering from CDK, the body weight of said animals is normalized, and this to a higher degree compared to felines which have not been treated with said primed MSCs or composition. Said improvement in/normalization of body weight of said feline can occur at any time point after administration of said primed MSCs or pharmaceutical composition comprising a therapeutically effective amount of said primed MSCs. In an embodiment, said improvement is measured 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years after administration of a first, second, third or further dose of said primed MSCs or pharmaceutical composition comprising a therapeutically effective amount of said primed MSCs. In an embodiment, said improvement is measured compared to felines which have been treated with unprimed/native MSCs. In an embodiment, said improvement is measured compared to felines which have been treated with a placebo solution. In an embodiment, said body weight is increased with at least 10 grams, preferably at least 20 grams, preferably at least 30 grams, preferably at least 40 grams, preferably at least 50 grams, preferably at least 60 grams, preferably at least 70 grams compared to baseline values measured before administration of said primed MSCs or pharmaceutical composition comprising a therapeutically effective amount of said primed MSCs.

The invention is further described by the following non-limiting examples which further illustrate the invention, and are not intended to, nor should they be interpreted to, limit the scope of the invention.

EXAMPLES AND/OR DESCRIPTION OF FIGURES

The present invention will now be further exemplified with reference to the following examples. The present invention is in no way limited to the given examples or to the embodiments presented in the figures.

Example 1: PGE₂ concentration evaluation in the media of primed ePB-MSCs

Set-up

For this experiment, three native equine Peripheral Blood (ePB)-MSC batches derived from three different donor horses (i.e. horse 1, horse 2 and horse 3) were primed with different cytokines (i.e. IL-6, IL-ip and TNF-o) for three consecutive days with a concentration of 10 ng/mL for each cytokine. Next, the cell culture medium was collected at the end of the cultivation period to assess the PGE₂ concentration using ELISA.

Results

The PGE₂ concentration was the lowest in the cell culture medium derived from the ePB-MSCs that were primed with IL-6 and the highest in the cell culture medium derived from the ePB-MSCs that were primed with a cytokine combination of TNF-o and IL-ip (see figures 1A and IB, showing fold change compared to PGE₂ concentration in the cell culture medium derived from ePB-MSCs from the same horse(s) that were not primed).

Example 2: IL-6 concentration evaluation in the media of primed ePB-MSCs Set-up

For this experiment, three native ePB-MSC batches derived from three different donor horses (i.e. horse 1, horse 2 and horse 3) were primed with a combination of two different cytokines (i.e. IL-13 and TNF-o) for three consecutive days with a concentration of 10 ng/mL for each cytokine. Next, the cell culture medium was collected at the end of the cultivation period to assess the IL-6 concentration using ELISA.

Results

The IL-6 concentration was two times higher in the cell culture medium derived from the ePB-MSCs that were primed with a cytokine combination of TNF-o and IL-13 (22.2 ± 1.9 pg/mL) when compared to native ePB-MSCs (11.4 ± 1.0 pg/mL) (see figure 2).

Example 3: Evaluation of efficacy and safety of native and primed ePB-MSCs in cats suffering from CKD.

Set-up:

The safety and efficacy of native and primed equine peripheral blood-derived mesenchymal stem cells (ePB-MSCs) was evaluated following an intravenous (IV) injection in cats suffering from chronic kidney disease stage 2 or 3. A third treatment group that received a placebo treatment was included.

Cells were primed by adding 10 ng/mL TNF-o and 10 ng/mL IL-ip. Seven cats were treated with native ePB-MSCs (in DMEM low glucose medium supplemented with 10% DMSO), six cats were treated with primed ePB-MSCs (in DMEM low glucose medium supplemented with 10% DMSO) and five cats were treated with placebo (saline). All cats were injected intravenously, independent of the type of treatment (native versus primed versus placebo). During the follow-up period of 12 weeks, a hematological and serum biochemistry analysis, urine analysis, general clinical assessment, quality of life assessment and injection site observation were performed. Quality of life was assessed based on the Quality of Life questionnaire derived from the validated questionnaire as reported by Bijsmans et al. Bijsmans ES, Jepson RE, Syme HM, Elliott J, Niessen SJ. Psychometric Validation of a General Health Quality of Life Tool for Cats Used to Compare Healthy Cats and Cats with Chronic Kidney Disease. J Vet Intern Med. 2016 Jan-Feb;30(l): 183-91. doi: 10.1111/jvim.13656. Epub 2015 Nov 14. PMID: 26567089; PMCID: PMC4913638.'). The systolic blood pressure and body weight were also recorded during the study period. All parameters were normalized to baseline values obtained on day 0. Results:

Seven cats were treated intravenously with the native ePB-MSCs, six cats were treated with primed ePB-MSCs and five cats were treated with saline (placebo group). All cats were evaluated for adverse events. No serious adverse events related to the native or primed ePB-MSCs, no suspected adverse drug reactions and no abnormal clinical signs were observed in the animals after IV injection. The IV injection of primed ePB-MSCs led to an increased quality of life over time in all groups, with a stronger and longer effect in both stem cell groups compared to placebo (see figure 3).

Furthermore, the cats treated with the (primed) ePB-MSCs showed a distinct decrease in systolic blood pressure values when compared to the placebo group, indicating a better progression (see figure 4). In addition, there was a more pronounced decrease in systolic blood pressure in the group treated with primed ePB-MSCs when compared to the group treated with native ePB-MSCs (Figure 4). The mean body weight of the cats treated with the primed epbMSCs showed an increase in comparison with the native ePB-MSC and placebo group (see figure 5).

The present invention is in no way limited to the embodiments described in the examples and/or shown in the figures. On the contrary, methods according to the present invention may be realized in many different ways without departing from the scope of the invention.

Claims

1. Primed mesenchymal stem cells (MSCs) or a pharmaceutical composition comprising a therapeutically effective amount of said primed MSCs for use in the treatment of chronic kidney disease in an affected feline, wherein said composition is administered to an affected feline and wherein prior to said administration the MSCs are primed by in vitro culturing said MSCs in an inducing cell medium comprising TNF-o.

2. Primed MSCs or a pharmaceutical composition comprising a therapeutically effective amount of said primed MSCs for use according to claim 1, wherein TNF-o is present in said inducing cell medium in a concentration between 1 ng/ml to 50 ng/ml, preferably between 5 ng/ml and 15 ng/ml.

3. Primed MSCs or a pharmaceutical composition comprising a therapeutically effective amount of said primed MSCs for use according to any of the previous claims, wherein said inducing cell medium further comprises interleukin IL-ip.

4. Primed MSCs or a pharmaceutical composition comprising a therapeutically effective amount of said primed MSCs for use according to claim 3, wherein IL-ip is present in said inducing cell medium in a concentration between 1 ng/ml to 50 ng/ml, preferably between 5 ng/ml and 15 ng/ml.

5. Primed MSCs or a pharmaceutical composition comprising a therapeutically effective amount of said primed MSCs for use according to any of the previous claims, wherein said MSCs are intravenously administered.

6. Primed MSCs or a pharmaceutical composition comprising a therapeutically effective amount of said primed MSCs for use according to any of the previous claims, wherein said MSCs are derived from blood, preferably peripheral blood.

7. Primed MSCs or a pharmaceutical composition comprising a therapeutically effective amount of said primed MSCs for use according to any of the previous claims, wherein said MSCs are allogeneic or xenogeneic MSCs, preferably xenogeneic MSCs.

8. Primed MSCs or a pharmaceutical composition comprising a therapeutically effective amount of said primed MSCs for use according to any of the previous claims, wherein said MSCs are mammal-derived, preferably equine- derived.

9. Primed MSCs or a pharmaceutical composition comprising a therapeutically effective amount of said primed MSCs for use according to any of the previous claims, wherein a dose of 1 x 10⁵ - l x 10⁷ MSCs per animal is administered.

10. Primed MSCs or a pharmaceutical composition comprising a therapeutically effective amount of said primed MSCs for use according to any of the previous claims, wherein a single dose is administered, or multiple doses are administered.

11. Primed MSCs or a pharmaceutical composition comprising a therapeutically effective amount of said primed MSCs for use according to any of the previous claims, wherein said primed MSCs have an increased secretion of the immunomodulatory prostaglandin E₂ cytokine.

12. Primed MSCs or a pharmaceutical composition comprising a therapeutically effective amount of said primed MSCs for use according to any of the previous claims, wherein said primed MSCs have an increased secretion of the immunomodulatory prostaglandin E₂ cytokine compared to unprimed MSCs, said increased secretion comprising a fold change of at least 2.

13. Primed MSCs or a pharmaceutical composition comprising a therapeutically effective amount of said primed MSCs for use according to any of the previous claims, wherein the creatinine levels in said felines are reduced and/or quality of life (QoL) scores are improved and/or blood pressure is normalized and/or the body weight is normalized, compared to a feline which has not been treated with said MSCs or composition.

14. A pharmaceutical composition comprising peripheral blood-derived MSCs, wherein said MSCs are mammal-derived, preferably equine-derived, and present in a sterile liquid at a concentration of between 1 x 10⁵ - l x 10⁷ MSCs per mL of said composition, wherein said composition has a volume of about 0.5 to 5 ml, wherein said MSCs measure positive for mesenchymal markers CD29, CD44 and CD90 and measure negative for MHC class II molecules and CD45, wherein said MSCs have a suspension diameter between 10 pm and 100 pm and wherein said MSCs are primed by means of an in vitro culturing step in an inducing cell medium comprising TNF-o.

15. Pharmaceutical composition according to claim 14, wherein said inducing cell medium further comprises interleukin (IL)-ip.

16. TNF-o primed MSCs, wherein said MSCs are preferably equine-derived and derived from blood, preferably peripheral blood.

17. Renal primed MSCs, wherein said MSCs are preferably equine-derived and derived from blood, preferably peripheral blood.