WO2017032612A1 - Stem cells with enhanced clonogenic, migratory and homing capacities - Google Patents

Abstract

The invention relates to stem cells with enhanced clonogenic, migratory and homing capacities and a method of obtaining said stem cells comprising co-culturing said stem cells in contact with at least a resting or activated NK cell. Also relates to compounds and compositions comprising said stem cells and their use for the treatment of diseases that may benefit from tissue regeneration, bone marrow transplantation and wound healing.

Description

Stem cells with enhanced clonogenic, migratory and homing capacities

The invention relates to the field of cell therapy and advanced therapies and more specifically to compounds, compositions and methods useful for the treatment of diseases that may benefit from tissue regeneration, bone marrow transplantation and wound healing. Also relates to the role of SDF-1 CXCR axis for promoting cell migration, homing, engraftment and regeneration. Diseases that may benefit from this invention are peripheral artery disease, myocardial infarction, stroke, erectile dysfunction, wound healing, hematopoeitic transplantation, skin engineering, diabetic neuropathy and others.

BACKGROUND OF THE INVENTION

Mesenchymal stromal cells (MSC) migration, homing and Engraftment

MSCs were first described by Friedenstein et al. (Friedenstein et al., 1974. Transplantation 17(4): 331-340; Friedenstein et al., 1974. Experimental Hematology 2 (2): 83-92, 1974) who described a population of cells derived from bone marrow that had the appearance of fibroblasts and may differentiate into bone, cartilage or adipose tissue, grew out as colonies ("colony forming units" or CFUs). Later on, Caplan and others termed these cells "mesenchymal stem cells" (MSCs) (Caplan1991 . Journal of Orthopaedic Research 9(5): 641-650; Prockop 1997. Science 276 (5309): 71-74). MSCs can be found in the bone marrow, adipose tissue, umbilical cord, placenta, among others. Since MSCs represent the adherent fraction of many tissue- derived cell cultures thay may be confused with fibroblasts. In the bone marrow they constitute the "stromal fraction" cells. MSC display characteristic cell surface antigen profile. The International Society for Cellular Therapies proposed a standardized phenotype. A typical human (h)MSC should express CD105, CD90, and CD73 but not CD79a, CD45, CD34, CD19, CD14, CD1 1 b. Homing of MSCs

One of the unsolved problems in cell-based therapies is the migration and integration of the cells to the site of injury, a process termed "homing." The therapeutic efficacy of MSCs is greatly dependent on their ability to produce juxtacrine or paracrine factors that enhance regeneration from endogenous (stem) cells. Several procedures have been suggested to increase migration and homing, such as fucosylation among others. Here we show that CB-NK (cord blood natural killer) cells increase cell migration, homing and engraftment of several cell types (progenitors, angiogenic, hematopoietic, among others). Immunological properties.

MSCs have extensive immunomodulatory and immunological tolerance inducing characteristics (Tse, et ai, 2003. Transplantation 75 (3): 389-397; Nicola et ai, 2002. Blood 99(10): 3838- 3843). Since hMSCs lack expression of MHC-II, CD40, CD80 and CD86 but express MHC-I, these cells are non-immunogenic. MSCs also efficiently suppress an immune response by modulating T-cell activation and proliferation. In fact, MSC have been efficiently used to prevent or to stop graft-versus-host disease (GVHD) (Le Blanc et ai, 2004. The Lancet 363(9419): 1439-1441 ), during organ transplantation or in the treatment of autoimmune diseases, such as type 1 diabetes mellitus, Crohn's disease and lupus among others. In addition, MSCs release trophic factors, anti-inflammatory cytokines or angiogenic factors.

MSCs are therefore currently evaluated in therapies for regenerate tissues and ischemic disorders, such as stroke, myocardial infarct, or peripheral arterial disease (PAD).

The SDF-1 (also termed CXCL12) CXCR4 axis

Stromal cell-derived factor-1 a (SDF-1 ) is a chemokine that plays a major role in cell trafficking and homing of CD34+ stem cells and other progenitor cells. Elevation of SDF-1 levels by cells of injured tissues leads to the recruitment of progenitors cells and stem cells, such as CD34+ cells, mesenchymal stromal cells, mesothelial cells, and others. The receptor for this chemokine is CXCR4, which was previously called LESTR or fusin. The CXCL12-CXCR4 interaction used to be considered exclusive (unlike for other chemokines and their receptors), but recently it was suggested that CXCL12 may also bind the CXCR7 receptor. The gene for CXCL12 is located in human chromosome 10. In human and mouse both CXCL12 and CXCR4 show high sequence identity: 99% and 90%, respectively. Many studies have shown therapeutic potential employing SDF-1/CXCR4 in tissue regeneration and engineering. SDF-1 -mediated homing via CXCR4 receptor in mesenchymal stem cell (MSC) effects as well as areas such as therapeutic angiogenesis, wound healing and neuronal and liver regeneration. The CXCR4 chemokine receptor that recognizes CXCL12 (stromal derived factor 1 alfa, also termed SDF-1 a) is highly expressed on bone marrow MSCs and others, but may be lost upon culturing. Between the factors that may increase homing are: culture of MSC with cytokines (such as HGF, SCF, IL-3, and IL-6); under hypoxic conditions, CXCR4 expression can be reestablished and expression of matrix metalloproteases (MMPs), among others. Homing is mostly dependent on the chemokine receptor CXCR4, and its binding partner that was previously characterized in HSC homing, that is, stromal-derived factor-1 CXCL12.

Angiogenesis in Peripheral Artery Disease and other Ischaemic processes Peripheral artery disease (PAD) constitutes the arterial stenosis of the lower or upper extremity, often secondary to atherosclerosis or thrombosis, but can also be due to other conditions such as embolitic disease, vasculitis, thomboangiitis obliterans, fibromuscular dysplasia, entrapment syndromes, endofibrosis, among others. The prevalence of PAD is expected to rise with the aging population, increase in obesity, type 2 diabetes, tobacco consumption, etc. Then, burden of PAD and associated cardiovascular and cerebrovascular morbidity and mortality continues to increase. Critical limb ischemia (CLI) represents the final stage of PAD and is characterized by rest pain, non-healing ulcers, and gangrene of the diseased leg. CLI is a major cause of decreased mobility, quality of life (QoL), and functional capacity, as well as an increased risk of amputation or death. As first-line therapies, patients with PAD are treated with a combination of risk-factor modification, antiplatelet drugs and statins. However, some patients fail to respond to first-line therapies or are not candidates for endovascular or surgical procedures and have no option other than amputation. Cell therapy-induced angiogenesis attempts to offer an alternative treatment for this subgroup of patients.

Currently, revascularisation, either surgical or endovascular, is the treatment cornerstone. However, this is not possible is several scenarios. In these "end-stage" patients, cell-based therapy has been identified as a potential new therapeutic option to induce therapeutic angiogenesis.

The most important outcomes of any intervention in patients with critical limb ischaemia are symptomatic relief, limb salvage, and functional improvement. The therapeutical approach for these patients requires a multidisciplinary care to control atherosclerotic risk factors, provide revascularization as far as possible, optimize wound care, adapt shoe wear, treat infection, and initiate rehabilitation therapy. Nevertheless, the cornerstone of the management is arterial reconstruction and limb salvage, which may lower impressively the rate of major amputation at one year, from 95% to 25%. Two methods are the mainstay of revascularisation: 1 ) open surgical techniques to remove the blockage or bypass it with vein or prosthetic graft or 2) endovascular treatment to reopen blocked arteries using a variety of catheter-based strategies that either crush or remove the blockage (balloon angioplasty, rotablation, atherectomy, etc.). However, despite advances in percutaneous and surgical revascularization techniques, up to 40% of patients are not candidates for revascularization at initial presentation (Norgren L and Group. 2007) This may occur when vascular obstruction involves a long segment, there is diffuse vascular disease, obstructions have distal location or there are very poor run off vessels. (Graziani L 2007) (O'Loughlin A 2010) So far the only pharmaceutical drugs with some positive (although divergent) results were prostanoids. Several other medical options have been tested, but none has shown long-term efficacy. Cell-based therapy is a novel and attractive potential treatment strategy for patients with critical limb ischemia, based on the facts that endothelial cells and hematopoietic stem cells derive from a common precursor, the hemangioblast and that under certain conditions patients may display a limited mobilization of endothelial progenitors from the bone marrow. This therapeutic option was suggested a decade ago, with the pioneer observation that CD34+ cells isolated from peripheral blood had the capacity to differentiate into fully differentiated and functional endothelial cells in culture. There have been multiple reports on the efficacy of cell transplantation in improving limb salvage in patients with critical limb ischaemia and various forms of therapeutic angiogenesis become a promising therapeutic option in these patients. The first landmark trial applying therapeutic angiogenesis using cell transplantation in patients with ischaemic limbs because of peripheral arterial disease was the TACT study. (Tateishi- Yuyama E The Lancet 2002). This trial, using intra-muscular implantation of autologous bone marrow-mononuclear cells, showed that in patients with critical limb ischemia not candidates for revascularisation cell transplant effectively increased blood flow in legs, as assessed by substantial increases in ankle-brachial index , transcutaneous oxygen pressure and pain-free walking time or by formation of new collateral vessels formation on angiogram. Benefits were maintained during at least 2 years after the therapy. More recently, Ruiz-Salmeron et al (Cell Transplantation 201 1 , 20(10): 1629-1639) the PROVASA Trial (Walter et al., 201 1 . Circ Cardiovasc Interv. 4:26-37) showed that intra-arterial administration of marrow-derived mononuclear cells improved ulcer healing and reduced rest pain, although a significantly increase ankle-brachial index was not achieved. Other studies showed not significant benefits beyond clinical evaluation. Firstly, there was a significant development of significant neovascularisation after cell therapy, as accessed by angiography, muscle biopsy or videocapillaroscopy. Additionally, a significant increase of perfusion evaluated by technetium- 99m-tetrofosmin scintigraphy was seen in the treated patients

Cell-based therapy is an attractive approach that has the potential to be applied in therapeutic angiogenesis. Animal data of hind-limb ischemia models and human trials of PAD done thus far have employed the transplantation of either autologous or allogeneic cells. Some of the cell sources under investigation include endothelial progenitor cells, bone marrow mononuclear cells, mesenchymal stem cells (bone marrow, adipose-derived), among others. Tissue regeneration by resident progenitors

Cell recruitment of resident progenitors to the damaged tissue, such as heart, brain or ischaemic legs is a major challenge to improve any cell based therapy. The process requires homing and engraftment of stem or progenitor cells. Major strategies to improve stem or progenitor cell homing used so far included the use granulocyte colony-stimulating factor (G- CSF), parathyroid hormone, statins, erythropoietin, and others. All these strategies are based on an improvement of stem or progenitor cell mobilization from the bone marrow. Strategies that have been shown to be successful are no major impact on hemodynamics was found in most of them. Then, improvement of stem cell homing is a major challenge in the development of successful cell based therapies but not yet improved to a clinical relevant status.

The importance of the chemokine receptor CXCR4 in HSC homing to the BM is well documented (Bonig et al., 2006. Blood Jan 1 ; 107(1 ): 79-86). Recent reports correlate CBSC migration in vitro with engraftment in humans, providing further support for the model that HSC migration is a critical step in the successful establishment of hematopoiesis (Voermans et al., 2001 . Blood 2001 Feb 1 ; 97(3): 799-804) .

Homing and Engraftment in Bone Marrow Transplantation

Hematopietic Cord blood transplantation (CBT) offers many advantages including rapid accessibility of the graft, less stringent HLA matching, a lower incidence of Graft versus Host Disease (GvHD) and a preserved Graft versus Leukemia (GvL) effect as compared to transplantation performed with bone marrow (BM) or mobilized peripheral blood (mPB) hematopoietic stem cells (HSC) (Broxmeyer et al., 2006. Methods in enzymology 419: 439- 473). However, some of the main limitations of CBT are a lower number of nucleated cells per cord blood (CB) unit, delayed engraftment and immune reconstitution and a higher incidence of infection. It has been described that the low HSC dose, the leukocyte content of the graft and ineffective CB CD34+ cells homing to the BM are factors associated with graft failure post-CBT. Recipients that require low HSC doses, such as pediatric patients, are the best candidates to receive CB grafts, however, adult patients who require a higher HSC dose represent a greater challenge for CBT. Double CBT, using two unmanipulated CB units, have been performed to increase the cell dose for adult patients. This approach results in better engraftment, however long-term engraftment is generally derived from one unit only. It has been described that the unit dominance is determined by CD34- accessory cells from the CB graft, suggesting that accessory cells participate in the engraftment process. Whilst several studies have shown the importance of accessory cells present in the graft for optimal HSC engraftment, a specific cell type that can be used to improve engraftment has yet to be identified. Recently, it was reported that the co-transplantation of umbilical cord-derived mesenchymal stem cells with the corresponding CB unit results in better engraftment as compared to patients transplanted with the CB graft only (Wu et al., 2013. Transplantation 2013 Mar 15; 95(5): 773-777). Other groups have reported a positive correlation between T cell dose and engraftment in adult patients post- CBT. In humanized mice, T cell depleted CB grafts showed inferior levels of engraftment, which could be enhanced when T cells stimulated ex vivo were co-infused. Similarly, a report using BM grafts showed that CD8+ T cells lacking cytotoxicity support initial HSC engraftment whereas CD8+ T cells with intact cytotoxic functions are needed to support long-term engraftment. Moreover, a higher graft content of cytotoxic cells, CD8+ T cells and NK cells, correlated with early engraftment and better outcome after transplantation with mPB HSC (Kim DH, Lee NY, Sohn SK, Suh JS, Lee KB. Non-CD34+ cells, especially CD8+ cytotoxic T cells and CD56+ natural killer cells, rather than CD34 cells, predict early engraftment and better transplantation outcomes in patients with hematologic malignancies after allogeneic peripheral stem cell transplantation. Biol Blood Marrow Transplant 2006; 12(7): 719-728). Lastly, improved levels of engraftment were reported in mice that received donor NK cells and IL-15 in a mouse model of non-myeloablative allogeneic BM transplantation and in patients following transplantation using a CD3/CD19 depleted graft.

CD34+ cells (HSC) must migrate to the BM in order to engraft and facilitate long-term immune reconstitution. It is known that the CXCR4/SDF-1 axis, LFA-1 (CD1 1 a), VLA-4 (CD29/CD49d) and VLA-5 (CD29/CD49e) all play key roles in HSC homing and maintenance within the BM niche n addition, it has been shown that CXCR7 may also be involved in this process through interaction with CXCR4. Thus, efforts have been made to enhance HSC engraftment by improving HSC homing. Recently, it was shown that inhibition of CD26, the dipeptidylpeptidase IV (DPPIV) enzyme that cleaves and inactivates SDF-1 a, results in enhanced migration of HSC in vitro and improved homing and engraftment of CBSC into lethally irradiated humanized mice. Likewise, HSC surface fucosylation improve CBSC homing and engraftment in vivo. Nevertheless, the effect of accessory cells on CBSC homing and engraftment requires further investigation.

Conclusion

A great deal of work has been done to harness the potential of stem cells for the treatment of patients. Cord Blood Stem Cells and Hematopoietic Stem cells used in bone marrow reconstitution, mesothelial cells as corneal endothelium surrogates. MSC and the likes are already undergoing clinical trials for use in patients especially for their immunomodulatory features. However, their heterogeneity and off-target homing especially lodging in the lungs limits the clinical use of MSC and MSC-like cells. Owing to this, a large number of cells are required to obtain desired effect at the target organ(s). Then, although use of stem cells would be highly beneficial in therapeutic applications, there is a need in the art to target cells to a desired location in the patient in need of such therapy in order to maximize their therapeutic potential.

SUMMARY OF THE INVENTION

This invention reports that cord blood natural killer cells (CB-NK) impact on progenitor and stem cells function by modifying their homing receptor repertoire, improving their migration and clonogenic capacities in vitro, and by enhancing homing and engraftment in vivo.

Therefore, a first aspect of the invention refers to a method of obtaining a stem cell with enhanced clonogenic capacity and migratory capacity, hereinafter first method of the invention, comprising co-culturing a stem cell with at least a NK cell. Preferably the NK cell is an activated NK cell, and more preferably, the NK cell has been activated with IL-15. In a more preferred embodiment, the NK cell is a cord blood natural killer cell (CB NK).

In another preferred embodiment, the stem cell is selected from the list consisting of mesenchymal stem cells, hematopoietic stem cells, mesothelial cells, embryonic stem cells, induced pluripotent stem cells, adult stem cells, hematopoietic stem cells, umbilical cord blood stem cells, or combinations thereof.

A second aspect of the invention relates to a stem cell obtainable by the first method of the invention, hereinafter, stem cell of the invention.

A third aspect relates to a stem cell population comprising at least one stem cell of the invention.

A fourth aspect of the invention relates to a composition, hereinafter composition of the invention, comprising: a) a cell co-culture comprising a stem cell, a NK cell and IL-15, b) a stem cell of the invention, or c) a stem cell population of the invention.

Preferably, the NK cell of step a) is a CB NK cell. A fith aspect of the invention relates to a kit of parts, hereinafter kit of parts of the invention, comprising: a) a stem cell, a NK cell and IL-15, or b) a stem cell and an IL-15 activated NK cell. Preferably, the NK cell of step a) is a CB NK cell.

A sixth aspect of the invention relates to the stem cell of the invention, the stem cell population of the invention, the composition of the invention, or the kit of parts of the invention, for use in therapy. A seventh aspect of the invention relates to the stem cell of the invention, the stem cell population of the invention, the composition of the invention, or the kit of parts of the invention, for use in partially or completely increase, restore or replace the functional activity of a diseased or damaged tissue or organ.

An eighth aspect of the present invention relates to the stem cell of the invention, the stem cell population of the invention, the composition of the invention, or the kit of parts of the invention, for use in the treatment, amelioration or prevention of a bone marrow disease or disorder.

In a preferred embodiment the bone marrow disease or disorder is selected from the list consisting of: a leukemia, a lymphoma, a myeloproliferative disease, a myelodysplasia syndrome and a plasma cell disorder, or any combinations thereof.

In another preferred embodiment, the leukemia is selected from the group consisting of Acute Lymphocytic Leukemia (ALL), Acute Myelocytic Leukemia (AML), Chronic Lymphocytic Leukemia (CLL), Chronic Myelocytic Leukemia (CML), Chronic Myelomonocytic Leukemia (CMML), stem cell leukemia, or any combinations thereof.

In another preferred embodiment the lymphoma is selected from the group consisting of Anaplastic Large-Cell Lymphoma (ALCL), Hodgkin's lymphoma, non-Hodgkin's lymphoma, or any combinations thereof. In another preferred embodiment the myelodysplastic syndrome is selected from the group consisting of Refractory anemia (RA), Refractory anemia with ringed sideroblasts (RARS), Refractory cytopenia with multilineage dysplasia (RCMD), Refractory cytopenia with multilineage dysplasia and ringed sideroblasts (RCMD-RS), Refractory anemia with excess blasts I and II; 5q- syndrome, myelodysplasia unclassifiable, or any combinations thereof. In another preferred embodiment the myeloproliferative disorder is selected from Polycythemia Vera (PV), Essential Thrombocythemia (ET), Chronic Idiopathic Myelofibrosis (MF), or any combinations thereof.

In another preferred embodiment the plasma cell disease is a plasma cell neoplasm selected from the group consisting of multiple myeloma, plasmacytoma, macroglobulinemia, monoclonal gammopathy of undetermined significance (MGUS), or any combinations thereof.

In another preferred embodiment the bone marrow disease or disorder is a hemoglobinopathy selected from the group consisting of sickle-cell disease and S a- thalassemia. A ninth aspect of the present invention relates to the stem cell of the invention, the stem cell population of the invention, the composition of the invention, or the kit of parts of the invention, for use in the treatment, amelioration or prevention of ischemia or peripheral artery disease.

In a preferred embodiment the ischemia or peripheral artery disease is selected from the list consisting of cerebrovascular ischemia, renal ischemia, pulmonary ischemia, limb ischemia, ischemic cardiomyopathy and myocardial ischemia or any combinations thereof.

In another preferred embodiment the ischemia or peripheral artery disease is critical limb ischemia.

A tenth aspect of the invention relates to the stem cell of the invention, the stem cell population of the invention, the composition of the invention, or the kit of parts of the invention, for use in the treatment, amelioration or prevention of myocardial infarction.

An eleventh aspect of the invention relates to the stem cell of the invention, the stem cell population of the invention, the composition of the invention, or the kit of parts of the invention, for use in the treatment, amelioration or prevention of stroke.

A twelfth aspect of the invention relates to the stem cell of the invention, the stem cell population of the invention, the composition of the invention, or the kit of parts of the invention, for use in the treatment, amelioration or prevention of erectile dysfunction.

A thirteenth aspect of the invention relates to the stem cell of the invention, the stem cell population of the invention, the composition of the invention, or the kit of parts of the invention, for use in the treatment, amelioration or prevention of wound healing. A fourteenth aspect of the invention relates to the stem cell of the invention, the stem cell population of the invention, the composition of the invention, or the kit of parts of the invention, for use in the treatment, amelioration or prevention of diabetic neuropathy. A fifteenth aspect of the invention relates to the stem cell of the invention, the stem cell population of the invention, the composition of the invention, or the kit of parts of the invention, for use in hematopoeitic transplantation.

A sixteenth aspect of the invention relates to a method of enhancing stem cell homing and engraftment potential, hereinafter second method of the invention, comprising the co-culture of said stem cell with at least a NK cell, preferably a CB-NK cell. Another aspect of the invention refers to the use of a NK cell to enhance stem cell homing and engrafment. Preferably the NK cell is an activated NK cell, and more preferably, the NK cell has been activated with IL-15. In a more preferred embodiment, the NK cell is a cord blood natural killer cell (CB-NK cell).

DESCRIPTION OF THE FIGURE

Figure 1 : CB-NK cells on cell migration and angiogenesis

The chemokine receptor CXCR4 is essential for migration and homing of bone marrow cella as well as circulating endothelial progenitors cells effects enhancing neovascularization after ischaemia. Effect of CB-NK cells on the angiogenic and migratory capacity of endothelial progenitor cells. A. Quantitative analysis of tube formation in a ex-vivo tube forming assay. Angiogenesis was measured by tube length in mm with a METAMORPH software (Ruiz Salmeron et al Cell Transplantation 201 1 , 20(10): 1629-1639). Data are mean ± SD (n = 3). B. Quantitication of basal migration of cultured EPC with or without CB-NK and antibody against CXCR4. Data are mean ± SD (n = 3)

Hindlimb ischaemia model was used to assess the role of CB-NK cells on the angiogenic effect of adipose-derived mesenchynal stromal cells (aMSC). aMSC, either alone or in combination with CB-NK cells were injected intrarterially through the postligation femoral artery or delivered intramuscularly.

Figure 2: CB-NK cells potentiate the effect of adipose-derived mesenchymal stromal cells on ischemic revascularization

After unilateral femoral ligation intramuscular delivery of adipose-derived mesenchymal stromal cells (alone) or plus resting CB-NK (aMSC+rCB-NK) or activated CB-NK cells (aMSC+aCB-NK). CB-NK cells accelerated the recovery of hindlimb blood flow as compared with controls or aMSC injected animals.

Figure 3: CB-NK and MSC cells on osteoporosis

A. Adipose-derived mesenchymal stromal cells from mouse. B. Differentiation into bone. C. Microcomputed tomography analysis of distal femur metastasis of sham, aMSC treated and aMSC + CB-NK treated mice. Data: mean ± SD (n=3)

Figure 4. Level of hCD45+ cell engraftment in the BM and fold increase in engraftment observed in NSG mice transplanted with CBSC and CB-NK cells.

(A) CD69 expression measured as mean fluorescence intensity (MFI)on CB-NK cells before and after incubation with 20ng/ml_ IL-15 for 4 h (N = 18). (B) Percentage of hCD45+cells detected by flow cytometry in the BM of NSG mice ten weeks post-transplant with CBSC alone or in combination withCD4+ T cells, CD8+ T cells, rNK cells or aNK (activated CB-NK) cells (N = 4). (C) Percentage of hCD45+cells detected by flow cytometry in the BM of NSG mice ten weeks post-transplant with CBSC alone or in combination with rNK (resting CB-NK) cells or aNK cells (activated CB-NK) (N =14). (D) Percentage of hCD45+ cells detected by flow cytometry in the BM of NSG mice transplanted with CBSC alone or in combination with rNK (resting CB-NK) cells or aNK (activated CB-NK) cells from 6 different CB units (N = 18).^* P < 0.05, ^** P < 0.01 , ^*** P < 0.001 .

Figure 5. Expression of homing receptors by CBSC following 4h co-culture with resting and activated CB-NK cells.

Results are expressed asMFI ± SD of positive cells for alpha integrins CD49d, CD49e, CD49f and CD1 1 a; beta integrins CD29 and beta-7; adhesion molecules CD162 and CD44 (A), and chemokine receptors CXCR4 and CXCR7 (B) (N =4). FMO samples were used as a negative control, ^* P < 0.05, ^** P < 0.01 , ^*** P < 0.001. Figure 6. Activated CBNK cells enhance CBSC migration capacity in vitro. CBSC migration capacity was analyzed following 4h transwell migration assays (N = 7). (A) Standard transwell migration assay using125 ng/mL SDF-1 a and 4 h incubation. (B) Suboptimal transwell migration assay using 50 ng/mL SDF-1 a and 3 h incubation. ^* P < 0.05.

Figure 7. Activated CB-NK cells enhance the short-term and long-term clonogenic capacity of CBSC. (A and B) Short-term CFU assays showing number of colonies formed after 4 h co- cultures of CBSC alone or with rNKcells (resting CB-NK) cells or aNK cells (activated CB-NK) (A) (N = 13). (C and D) Long-term 4 week cobblestone cultures followed by CFU assays (LTC- IC) showing number of colonies formed after 4 h co-culture of CBSC alone or with rNK cells (resting CB-NK) cells (C) or aNK cells (activated CB-NK) (D) (N = 8). (E) Cell cycle analysis by flow cytometry of CBSC alone or after co-culture with rNK cells (resting CB-NK) or aNK cells (activated CB-NK) . ^* P < 0.05, ^** P < 0.01.

Figure 8. Blocking integrin receptors on CBSC prevents activated NK cells from enhancing CBSC clonogenic capacity. CFU assays showing number of colonies formed after 4 h co- cultures of CBSC alone or with rNK cells or aNK cells. CBSC were blocked for 1 h prior to co- culture with antibodies against beta-7 integrin, CD1 1 a, CD49d, CD49e alone or in combination at 10 g/mL, in comparison to isotype control (N = 6), ^* P < 0.05, ^** P < 0.01.

Figure 9. Changes in gene and protein expression induced in CBSC after culture with resting or activated CB-NK cells. (A and B) CBSC were cultured for 4 h either alone or with resting or activated CB NK cells and then CBSC were re-isolated by cell sorting (N = 3). Gene expression profiles were compared by microarray analysis using GeneChip human Gene 2.0 ST Arrays and normalized. Data is presented as a Venn diagram and Volcano plots comparing the number of CBSC genes for which expression was changed (either up or down-regulated) by greater than 1 .5 fold (p < 0.05) after culture with resting or activated CBNK cells in comparison to CBSC alone (untreated). (C) Validation of microarray data showing increased CXCL-9 expression by CBSC after culture with activated CB NK cells. CXCL-9 protein was measured in culture supernatants using ELISA after co-culture of CBSC alone or with resting or activated CB NK cells (N = 4). (D) CFU assays showing number of colonies formed after 4 h culture of CBSC with different concentrations of CXCL9 as indicated (N = 4), ^* P < 0.05. Figure 10. Changes in gene and protein expression in NK cells after culture with IL-15. (A) Gene expression profiles were compared by microarray analysis using GeneChip human Gene 2.0 ST Arrays and normalized. Data is presented as a Volcano plot comparing the number of genes for which expression was changed (either up or down-regulated) by greater than 1 .5 fold (p < 0.05) between rNK cells and aNK cells. Refer to Table S2 for specific changes in gene expression. (B) IFN-γ and TNF-β secretion by NK cells was measured by ELISA following activation with IL-15 (N =24) to confirm microarray data, ^*** P < 0.001.

DESCRIPTION OF THE INVENTION At first, the authors of the present invention found that when cord blood stem cells (CB-SC) were co-cultured with NK cells activated with IL-15 (activated NK cells) (aNK cells) for 4 hours previous to transplantation, the increase in MFI (mean fluorescent intensity) expression of CXCR-4 compared to a control of CB-SC only and CB-SC co-cultured with non activated NK cells (rNK cells). Also, they found a higher number of CFUs when CB-SC were co-cultured with aNK cells compared to rNK cells (P < 0.05). In addition, the authors of the present invention performed transwell migration assays to analyse the effect of aNK cells on the migration capacity of CBSC, and surprisingly found that CB-NK cells play an important role in stem cells migration and engraftment. This previously unknown effect of CB-NK cells resulted in a significant potentiation of the beneficial effects of stem cells, progenitors cells and MSC. In the present invention, the inventors describe a method of enhancing migration, homing, adhesion, or engraftment of a cell such as a stem cell to an injured tissue or organ. This method is carried-out by contacting a stem cell with activated NK cells preferably with activated CB-NK cells (aCB-NK cells). METHOD OF OBTAINING A STEM CELL WITH ENHANCED CLONOGENIC CAPACITY AND MIGRATORY CAPACITY

Therefore, a first aspect of the invention relates to a method of obtaining a cell, or a cell population, with enhanced clonogenic capacity and migratory capacity, from hereinafter first method of the invention, comprising co-culturing a cell with a NK cell. Preferably, the cell is a stem cell. More preferably, the NK cell is an activated NK cell, still more preferably the NK cell has been activated with IL-15, and still more preferably the NK cell is a cord blood natural killer cell (CB-NK cell). There are several options in the literature in order to activate NK cells, including without limitation, pre-culture with K562 cells (Imai et al. 2005. Blood 106: 376-83), add IL-2 (Tse- Kuan Yu et al., 2000. J Immunol 164:6244-6251 ) or IL-15 (Allavena et al., 1997. J Leukoc Biol. Jun 61 (6): 729-735), or antibodies against NKG2D or NKp46 (Cox et al Eur J Immunol 2015 May 20. DOI: 10.1002/eji. 201444990). These different methods activate different pathways and have different effects. The authors of the present invention have shown herein that activation of naive CB-NK cells with IL-15 is the most appropriate method to achieve the technical effect of the present invention, namely for enhancing migration, homing, adhesion, or engraftment of a cell such as a stem cell to an injured tissue or organ. The examples of the invention show that NK cells were activated with IL-15 for 4 hours previous to transplantation. Therefore, preferably, the NK cell has been activated with IL-15 for at least 2 hours, preferably 3 hours, and more preferably for at least 4 hours.

The level of NK cell activation with IL-15 is variable and it is possible that a longer incubation time with IL-15 could lead to a more consistent activation of NK cells, preferably CB-NK cells. Interleukin 15 (I L-15) is a cytokine with structural similarity to IL-2. Like IL-2, IL-15 binds to and signals through a complex composed of IL-2/IL-15 receptor beta chain (CD122) and the common gamma chain (gamma-C, CD132). The period of time for the co-culture is variable according to the desired level of activation. In a preferred embodiment the stem cell is co-cultured with the activated NK cell for at least 4 hours, preferably for at least 12 hours, and more preferably for at least 24 hours. In a more preferred embodiment the NK cell is a CB-NK cell. The co-culture could be peformed either with NK cells previouslu activated with IL-15, or with NK cells activated during the culture. Therefore, in another preferred embodiment of the invention, the stem cell is co-cultured with the NK cell in presence of IL-15. In a more preferred embodiment the NK cell is a CB NK cell.

The method of enhancing homing and engraftment of a cell, may comprise providing one or more cells selected from stem cells, progenitor cells, neutrophils, macrophages and T-cells. The stem or progenitor cells may be embryonic stem cells, adult stem cells, expanded stem cells, placental stem cells, bone marrow stem cells, amniotic fluid stem cells, neuronal stem cells, cardiomyocyte stem cells, placental stem cells, endothelial progenitor cells, circulating and mobilized peripheral blood stem cells, muscle stem cells, germinal stem cells, adipose tissue derived stem cells, exfoliated teeth derived stem cells, hair follicle stem cells, dermal stem cells, parthenogenically derived stem cells, reprogrammed stem cells such as induced pluripotent stem cells or somatic nuclear transfer, or combinations thereof. One or more cells may be contacted with the activated CB-NK cell, enhancing homing and engraftment of this cell. This may result in a population of modified cells.

In another preferred embodiment the stem cells with enhanced clonogenic capacity and/or with enhanced migratory capacity could be selected from the list consisting of bone marrow mononuclear cells, CD34+ cells, CD 133+ cells, mesothelial cells, coord blood stem cells and/or mesenchymal stromal cells. In another more preferred embodiment, the stem cell is an umbilical cord blood stem cell (CBSC).

ISOLATED STEM CELL AND ISOLATED POPULATION STEM CELL OF THE INVENTION.

A second aspect of the invention relates to a cell obtainable by the method of the invention, hereinafter stem cell of the invention. Said cell has enhanced clonogenic capacity and/or migratory capacity. Preferably, said cell is a stem cell. In another preferred embodiment, the cell is selected from stem cells, progenitor cells, neutrophils, macrophages and T-cells. The stem or progenitor cells may be embryonic stem cells, adult stem cells, expanded stem cells, placental stem cells, bone marrow stem cells, amniotic fluid stem cells, neuronal stem cells, cardiomyocyte stem cells, placental stem cells, endothelial progenitor cells, circulating and mobilized peripheral blood stem cells, muscle stem cells, germinal stem cells, adipose tissue derived stem cells, exfoliated teeth derived stem cells, hair follicle stem cells, dermal stem cells, parthenogenically derived stem cells, reprogrammed stem cells such as induced pluripotent stem cells or somatic nuclear transfer, or any combinations thereof.

The term "stem cell" is selected from the list consisting of mesenchymal stem cells, hematopoietic stem cells, embryonic stem cells, induced pluripotent stem cells, adult stem cells, mesothelial cells, hematopoietic stem cells, bone marrow mononuclear cells, CD34+ cells, CD 133+ cells, mesothelial cells, umbilical cord blood stem cells, or combinations thereof.

In another preferred embodiment, the stem cell is an isolated stem cell. In another preferred embodiment, the stem cell is an adult stem cell.

By "adult" it is meant that the stem cells are not embryonic. In one embodiment, "adult" means post-embryonic or "post-natal". With respect to the stem cells of the present invention, the term "adult stem cell" means that the stem cell is isolated from a tissue or organ of an animal at a stage of growth later than the embryonic stage. In one aspect, the stem cells of the invention may be isolated at the post-natal stage such as the umbilical cord and placenta. The cells may be isolated from a mammal, such as a rat, mouse, pig or human. Adult stem cells are unlike embryonic stem cells, which are defined by their origin, the inner cell mass of the blastocyst. Adult stem cells according to the invention may be isolated from any non-embryonic tissue, and will include neonates, juveniles, adolescents and adult subjects. Generally the stem cell of the present invention will be isolated from a non-neonate mammal, and for example from a non- neonate human, rat, mouse or pig. Preferably, the stem cells of the present invention are isolated from a human, and are therefore human adult stem cells or a substantially pure population of human adult stem cells.

By Cord Blood Stem Cells (CB-SC) it is meant that NK cells were isolated from postnatal fetal blood remaining in the umbilical cord and placenta. aNK and rNK refer to activated, preferably with IL-15, (aNK) and resting (rNK) naive Cord Blood NK cells (CB-NK). In another preferred embodiment, said cell populations are expanded after co-culture with NK cells that has been activated previously with IL-15. In another preferred embodiment, said cell populations are not expanded after the co-culture. The authors of the present invention assessed whether CB-NK cells affect the homing properties of CD34+ cells by analyzing the expression of key adhesion molecules and chemokine receptors for homing to the bone marrow (BM). Receptor expression by CB-SC was assessed by flow cytometry following 4 hrs co-culture with rNK or aNK cells. Importantly, co- culture had no effect on CB-SC or NK cell viability. The inventors found that CB-SC expressed significantly lower levels of CD1 1 a (p < 0.05), CD29(p < 0.01 ), CD44 (p < 0.05) and integrin beta-7 (p < 0.05) after co-culture with aNK cells but not with rNK cells (Figure 5A). Interestingly, they observed significantly higher expression levels of the chemokine receptors CXCR4 (p < 0.001 ) and CXCR7(p < 0.05) on CB-SC co-cultured with aNK cells compared to CBSC alone or co-cultured with rNK cells (Figure 5B).

Consequently, in a preferred embodiment the stem cells of the invention express higher levels of the chemokine receptors CXCR4 and/or CXCR7 compared with other stem cells. In other preferred embodiment the stem cells of the invention express significantly lower levels of the markers selected from CD1 1 a, CD29, CD44, integrin beta-7, and/or their combinations.

The phenotypic markers of the stem cells of the invention can be identified by any suitable technique, usually based on positive/negative selection. In a particular embodiment, antibodies can be used, preferably monoclonal antibodies, to said phenotypic markers whose presence or absence in the stem cells of the invention must be confirmed with the aim of characterizing the stem cells of the invention on the basis of their immunocytochemical profile, although other conventional techniques known by a person skilled in the art can also be used, for example, RT- PCR.

As an increased expression of CXCR4 and CXCR7 by CB-SC was observed after co-culture with aNK cells, the inventors studied whether this led to an increased response of CB-SC to SDF-1 a in vitro. Migration of CB-SC in vitro towards SDF-1 a was significantly enhanced in the presence of aNK cells when compared to CB-SC alone (P < 0.05) (Figure 6A). Though an increased chemotactic index was observed in the presence of rNK cells, it was not significant when compared to CB-SC alone. In addition, the inventors performed transwell migration assays using a sub-optimal dose of SDF-1 a and a shorter incubation time. In line with the results obtained using a standard concentration of SDF-1 a, aNK cells and not rNK cells specifically enhanced the migration capacity of CB-SC towards a sub-optimal dose of SDF-1 a (P < 0.05) (Figure 6B). Therefore, in another preferred embodiment, the stem cell of the invention has enhanced migratory capacity. In this context, the term "stem cell" refers to a clonogenic cell with capacity for self-renewal and differentiation into multiple cell lineages. In particular, mesenchymal stem cells have the ability to extensively proliferate and form colonies of fibroblast cells. As used herein, the term "stem cell" refers to a totipotent, pluripotent or multipotent cell, capable of generating one or more differentiated cell types, and also has the ability to regenerate itself, ie to produce more stem cells. The "totipotent stem cell" can develope either the embryonic components (such as the three embryonic layers, the germ line and give rise to tissues yolk sac) and the extraembryonic (such as placenta). That is, they can form all cell types and lead to a complete organism. The "pluripotent stem cells" can form any cell type of the three embryonic lineages (endoderm, ectoderm and mesoderm), as the germinal and yolk sac. They can therefore form cell lineages from them but they cannot form a complete organism. The "multipotent stem cells" are those that can only generate cells of the same lineage layer or embryonic origin. Bone marrow contains at least two different populations of stem cells: mesenchymal stem cells (MSCs) and hematopoietic stem cells (HSCs). In this context of the present invention, stem cells are selected from the group comprising mesenchymal stem cells, hematopoietic stem cells, embryonic stem cells, induced pluripotent stem cells, adult stem cells, or combinations thereof. In a particular embodiment, the stem cells are stem cells from a mammal, preferably human. In a particular embodiment, the stem cells are mesenchymal stem cells, preferably human mesenchymal stem cells.

In addition, in this context the term "adult stem cell" refers to a stem cell that is isolated from a tissue or an organ of an animal in a state of post-embryonic growth. Adult" means post- embryonic. Preferably, the stem cells of the invention are isolated on a postnatal state. Preferably they are isolated from a mammal, and most preferably a human, including neonates, juveniles, adolescents and adults. They can be isolated adult stem cells of a variety of tissues and organs, such as bone marrow (mesenchymal stem cells, multipotent adult progenitor cells and hematopoietic stem cells), adipose tissue, cartilage, epidermis, hair follicle, skeletal muscle, heart muscle, intestine, liver, neuronal.

The term "embryonic stem cell" or "ESC" refers to cells derived from the inner cell mass of blastocyst stage embryos, capable of self-renewal and differentiation into all types of adult cells. Embryonic stem cells are able to proliferate indefinitely in vitro maintained in an undifferentiated state and normal karyotype through prolonged culture. They also have the ability to differentiate into cells from all three embryonic germ layers (mesoderm, endoderm and ectoderm; (Itskovitz- Eldor et al., 2000. Mol Med 6: 88-95) and the germ line. Embryonic stem cells represent a model of powerful system for investigating the mechanisms underlying pluripotent cell biology and differentiation in the early embryo, and provide opportunities for genetic manipulation. Embryonic stem cells have been isolated from the MCI blastocyst stage embryos in multiple species (Bhattacharya et al., 2005. BMC Dev Biol 5:22), including mice (Solter and Knowles, 1978. Proc Natl Acad USA 75:5565-5569), pig (Chen et al., 1999. Theriogenology. 52: 195- 212), non-human primates (Thomson et al., 1995. Proc Natl Acad USA 92, 7844-7848) and humans (Reubinoff et al., 2000. Nat Biotechnol 18: 399-404; Mandal et al., 2006. Differentiation 74: 81 -90).

Preferably, the invention contemplates the use of embryonic stem cells from established cell lines of human origin including without limitation ACT-14, AS034, AS034.1 , AS034.2, AS038, AS079, AS094, BGOI, BG02, BG03, BG04 lines Choi, CH02, CLSI, CLS2, CLS3, CLS4, ESOL, ES02, ES03, ES04, ES05, ES06, ESMOI, ESM02, ESM03, FC018, FES 21 FES 22 FES 29 FES 30, Geol, GE07, GE09 , GE13, GE14, GE91 , GE92, hES-NCLL, HS181 , HS207, HUESI, HUESI0, HUES1 1 , HUES12, HUES13, HUES14, HUES15, HUES16, HUES17, HUES2, HUES3, HUES4, HUES5, HUES6, HUES7, HUES8, HUES9 , H9, Ches-I, Ches-2, Ches-3, Mbol, MB02, MB03, LSAT-Miz, Miz-hES10, LSAT Miz-1 -hES12 Miz, Miz-LSAT 3 Miz- hES14, Miz-LSAT 5-hES2 Miz, Miz-hES3, hES4-Miz, Miz-Hes5, Miz-Hes6, Miz- Hes7, Miz-hES8, nCol, NC02, NC03, ReliCellhESI, RHL, RH3, RH4, RH5, RH6, RH7, RL05, RL07, RLIO, RL13, RL15, RL20, RL21 , Royan HI, SAOOI, SA002, SA002.5, SA046, SA085, SA1 1 1 , SA121 , SA142, SA167, SA181 , SA191 , SA196, SA202, SA203, SA21 1 , SA218 , SA240, SA279, SA348, SA352, SA399, SA61 1 , SI-100, SI-101 , SI-102, SI-103, SI-104, SI-105, SM 06, SI-107, SI-108, SI 109, SI-1 10, SI-1 1 1 , SM 14, SM 15, SM22, SI-123, SI-124, SI-125, SI-126, SM28, SI-130, Sl- 131 , SI-132, SI 133, SI-134, SM35, SI-137, SI-138, SM39, SI-140, SM41 , SI-144, SI-145, Sl- 146, SM48, SI-149, SI-15, SI-150, SI-151 , SI-153, SI-154, SI-155, SI-156, SM57, SI-158, Sl- 159, SM60, SM61 , SI-162, SI-163, SI-164, SI-165, SI-167, SI-168, SI-169, SI-170, SI-171 , Sl- 172, SI-174, SI-175, SI-176, SI-177, SI-178, SI-179, SI 18, SI-180, SI-182, SI-183, SI-184, Sl- 185, SI-186, SI-187, SI-188, SI-189, SI-191 , SI-192, SI-193, SI-194, SI-195, SI-196, SI-197, Sl- 198, SI-199, SI-200, SI-201 , SI-202, SI-203, SI-204, SI-205, SI 206, SI-208, SI-209, SI-21 , Sl- 210, SI-21 1 , SI-213, SI-214, SI-215, SI-216, SI-217, SI-221 , SI-24, SI-27, SI-28, SI-31 , SI-33, SI-53, SI-60, SI-62, SI-63, SI-79, SI-80, SI-81 , SI-93, SI 94, SI-95, SI-96, SI-97, SI-98, SI-99, SNUhESI, SNUhES2, SNUhES3, TE-03, TE-04, TE-06, TE-07, TE-32, TE 33, TE-62, TE-72, UCOL, UC06, VAL-I, VAL-2, Val-3, Val-4, WAOL, WA07, WA09, WA13 or WA14, all of which are available from public repositories. The invention contemplates the use of embryonic stem cells from established cell lines of murine origin such as 59B5, 36.5, 9TR#1 , TK#1 , ES-D3 [D3] lines, YS001 , ES-E14TG2a, ES- D3, 10p12, 56B3, L Wnt-3A, OP9, WT MEFs 3T3, 3T3 KO MEFs, 127TAg, 151TAg, WPE-stem, NE-4C, NE-GFP-4C, ES-C57BL / 6, J1 , R1 , RW.4 , B6 / BLU, SCC # 10, EDJ # 22, AB2.2, Ainv15, 7AC5 / EYFP, R1 / E, G-Olig2, CE-1 , CE3, and hESC BGOIV all of which are available on public repositories.

Methods for obtaining embryonic stem cells are widely known and can be implemented by the skilled person in the art without undue experimentation. Thus, human ES cells can be obtained as described in Reprod. Biomed. Online 4 (2002), 58-63. Primate embryonic cells can be isolated from blastocysts of different primate species (Thomson et al., 1995. Proc Natl Acad Sci USA, 92: 7844— 7848). Embryonic germ cells can be prepared from primordial germ cells present in human fetuses 8-1 1 weeks after the last menstrual period using methods such as described by Shamblott et al., 1998. Proc. Natl. Acad. Sci. USA 95 (23): 13726-31 .

In order to avoid the use of human embryos it is possible to use transgenic animals as a source of embryonic stem cells. Particularly US5.523.226 discloses methods for generating transgenic pigs that can be used as donors for xenotransplantation into humans. WO97/12035 describes methods of producing transgenic animals suitable for xenotransplantation. Also, WO01/88096 describes immuno animal tissues. These immuno animals can be used to generate pluripotent embryonic cells as described in US6.545.199.

It is also possible to use embryonic stem cell lines, which can be of different origin. In one embodiment, cell lines are mouse and include cells such as the R1 line (ATCC No. SCRC- 101 1 ) described by Nagy et al (Proc.Natl.Acad.Sci. USA, 1993, 90.: 8424-8428) and the cell line D3.

The term "hematopoietic stem cell" or "HSC" refers to an adult stem cell capable of giving rise to both hematopoietic lineages myeloid (monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes/platelets, dendritic cells) as lymphoid (T cells, B cells, NK cells). This cell type is found primarily in the bone marrow.

The term "mesenchymal stem cell" or "MSC", as used herein, refers to a multipotent stromal cell, arising from the mesodermal germ layer which can differentiate into a variety of cell types, including osteocytes (bone cells), chondrocytes (cartilage cells) and adipocytes (fat cells). Preferably, the markers expressed by mesenchymal stem cells include CD105 (SH2), CD73 (SH3 / 4), CD44, CD90 (Thy-1 ), CD71 and Stro-1 and adhesion molecules CD106, CD166, and CD29. The negative markers for MSCs (not specified) are the hematopoietic markers CD45, CD34, CD14, and costimulatory molecules CD80 , CD86 and CD40 as well as the adhesion molecule CD31 . MSCs may be obtained from, without being limited to, bone marrow, adipose tissue (such as subcutaneous fat), liver, spleen, testes, menstrual blood, amniotic fluid, pancreatic, periosteum, synovium, skeletal muscle, dermis, pericytes, trabecular bone, human umbilical cord, lung, peripheral blood and dental pulp. The MSC according to the invention may be obtained from any of the above tissues such as from bone marrow, subcutaneous adipose tissue or umbilical cord. MSC may be isolated from bone marrow by methods known to the skilled in the art. In general, such methods include isolation of mononuclear cells by density gradient centrifugation (Ficoll, Percoll) of bone marrow aspirates, and then seeding the cells isolated in tissue culture plates in a medium containing fetal bovine serum. These methods rely on the ability of MSCs to adhere to the plastic, so that while non-adherent cells are removed from culture, adherent MSC may spread on culture plates. The MSC also can be isolated from subcutaneous adipose tissue following a similar procedure known to the skilled in the art. A method of isolating bone marrow MSCs or subcutaneous adipose tissue has been previously described (De la Fuente et al, 2004. Exp Cell Res, Vol 297: 313- 328). In a particular embodiment of the invention, mesenchymal stem cells are obtained from umbilical cord, preferably human umbilical cord.

The term "mesothelial cells" refers to a type of simple squamous epithelial cells lining the walls of coelomic body cavities and visceral organs located inside. Biostructurally, the mesothelium represents a semi-permeable laminar interface that separate fluid-filled body cavities from blood vessels and lymphatics running within the underneath submesothelial connective tissue layers. Besides its property of physical barrier, the mesothelium acts also as bioactive interface regulating fluid flows interchanges across it surface to maintain an optimal osmolarity and ionic activity of body cavity fluids. These functions are mainly accomplished by transmembrane ion pumps (Na7K⁺-ATPase) and water channels (aquaporins). Mesothelial cells also actively regulate coelomic cavities homeostasis and inflammatory status via their secretion of numerous pro- and anti-inflammatory cytokines.lt has also been evidenced that mesothelial cells are actively recruited during serosal regeneration, through processes of proliferation, migration or delamination and secretion of a large variety of growth factors or cytokines. Their "stem cell" properties have been demonstrated recently by Lachaud et al (2013, 2014) Lachaud et al Cell Death Dis 5, e1304. doi: 10.1038/cddis.2014.271 ; Lachaud et al, 2013. PLoS One 8, e55181. doi: 10.1371/journal.pone.0055181 ; Lachaud et al, 2014, Invest Ophthalmol Vis Sci 55, 5967- 5978. doi: 10.1 167/iovs.14-14706).

The terms "pluripotent stem cells" or "pluripotent stem cell" and grammatical equivalents are used interchangeably in the context of the present invention to refer to undifferentiated or poorly differentiated cells, of any kind, able to divide indefinitely without losing their properties and capable of forming any cell of the three embryonic lineages (mesoderm, endoderm, ectoderm) and germ line and the germ line when grown under certain conditions. The invention contemplates the use of any type of pluripotent stem cell which is able to generate a progeny of any of the three germ layers including cells derived from embryonic tissue, fetal tissue, adult tissue and other sources. Suitable for use in the present invention include pluripotent embryonic stem cells, embryonic carcinoma cells, induced pluripotent stem (iPS) and primordial germ cells. The invention also contemplates the use of pluripotent stem cells from any species including, without limitation, human cells, mouse, rat, bovine, sheep, hamster, pig and the like. The term "induced pluripotent stem cell" or "iPS", as used herein, refers to cells which are substantially genetically identical to a differentiated somatic cell from which derive but show similar characteristics in terms of pluripotency and proliferative ability to the embryonic stem cells. Typically, iPS expressed surface markers selected from the group consisting of SSEA-3, SSEA-4, TRA-I -60, TRA-1 -81 , TRA-2-49/6E, and Nanog. Typically, iPS express one or more genes selected from the group of Oct-3/4, Sox2, Nanog, GDF3, Rexl, FGF4, ESGI, DPP A2, A4 and hTERT DPP. IPSs can be generated using methods described in the prior art such as the methods described by Takahashi and Yamanaka (Cell, 2006, 126: 663-676), Yamanaka et al. (Nature, 2007, 448: 313-7), Wernig et al. (Nature, 2007, 448: 318-24), Maherali (Cell Stem Cell, 2007, 1 : 55-70); Maherali and Hochedlinger (Cell Stem Cell, 2008, 3: 595-605), Park et al. (Cell, 2008, 134: 1 -10); Dimos et al. (Science, 2008, 321 : 1218-1221 ), Blelloch et al. (Cell Stem Cell,

2007, 1 : 245-247); Stadtfeld et al. (Science, 2008, 322: 945-949) and Okita et al. (Science,

2008, 322: 949-953). They are reprogrammed in vitro from somatic cells differentiated terminally by retroviral transduction of the transcription factors Oct3 / 4, Sox2, Klf4 and c-Myc cells. Typically, iPS cells are obtained from somatic cell by expressing in said cells of Oct- 3/4 and Sox2 protein of Oct-3/4, Sox2 and Klf4 proteins, protein Oct-3/4, Sox2, Klf4 and c-Myc and/or Oct-4, Sox2, Nanog and Lin28 protein.

If desired, the stem cell of the invention can be modified genetically by any conventional method including, by way of illustration but non-limiting, processes of transgenesis, deletions or insertions in their genome, etc.

The stem cells, progenitor cells or differentiated cells of the invention, the immortalized cells of the invention, as well as the cells present in the cell population of the invention, can be cells of autologous, allogeneic or xenogeneic origin. In a particular embodiment, said cells are of autologous origin and are isolated from the CB of the subject to whom they will be administered, thus reducing potential complications associated with antigenic and/or immunogenic responses to said cells. When donors are used to obtain the stem cells, the immune response can be minimized by matching the haplotypes of the donors to those of the recipients. In certain aspects of the invention, the stem cell population is considered not to trigger an immune response if at least about 70% of the cells of the isolated stem cell population do not trigger an immune response. In some embodiments, at least about 80%, at least about 90% or at least about 95%, 99% or more of the cells of the isolated stem cell population do not trigger an immune response. Preferably the cells of the invention do not trigger an antibody mediated immune response and/or do not trigger a humoral immune response and/or do not trigger a mixed lymphocyte immune response. If desired, the stem cells of the invention can be expanded clonally using a method that is suitable for cloning cell populations. Alternatively, a population of stem cells of the invention can be collected and the cells can be placed on separate plates (or in the wells of a multiwell plate). In another alternative embodiment, said stem cells can be subcloned on a multiwell plate in a random relation to facilitate the operation of placing a single cell in each well (e.g. from approximately 0.1 to about 1 cell/well). Of course, the stem cells of the invention can be cloned at low density (e.g. in a Petri dish) and can be isolated from other cells using suitable devices (e.g. cloning rings). The clonal population can be expanded in a suitable culture medium.

A third aspect relates to a stem cell population, hereinafter stem cell population of the invention, comprising at least one stem cell of the invention. More preferably, the stem cell population is substantially pure. Preferably, said population of the invention is a cell population in which at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, of the cells are stem cells of the invention, with enhanced clonogenic capacity and/or migratory capacity. In other words, in some embodiments at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, of the cells in the populaition are stem cells of the invention, with enhanced clonogenic capacity and/or migratory capacity.

The term "isolated" indicates that the cell or cell population to which it refers is not within its natural environment. The cell or cell population has been substantially separated from surrounding tissue. In some embodiments, the cell or cell population is substantially separated from surrounding tissue if the sample contains at least about 75%, in some embodiments at least about 85%, in some embodiments at least about 90%, and in some embodiments at least about 95% adult stem cells. In other words, the sample is substantially separated from the surrounding tissue if the sample contains less than about 25%, in some embodiments less than about 15%, and in some embodiments less than about 5% of materials other than the adult stem cells. Such percentage values refer to percentage by weight or by cell number. The term encompasses cells which have been removed from the organism from which they originated, and exist in culture. The term also encompasses cells which have been removed from the organism from which they originated, and subsequently re-inserted into an organism. The organism which contains the re-inserted cells may be the same organism from which the cells were removed, or it may be a different organism, i.e. a different individual of the same species. Cells of the invention are positive for certain phenotypic markers and negative for others. By "positive", it is meant that a marker is expressed within a cell. In order to be considered as being expressed, a marker must be present at a detectable level. By "detectable level" is meant that the marker can be detected using one of the standard laboratory methodologies such as PCR, blotting or FACS analysis. A gene is considered to be expressed by a cell of the population of the invention if expression can be reasonably detected after 30 PCR cycles, which corresponds to an expression level in the cell of at least about 100 copies per cell. The terms "express" and "expression" have corresponding meanings. At an expression level below this threshold, a marker is considered not to be expressed and the cell is then described as negative for this marker. A marker is considered not to be expressed by a cell of the invention, if expression cannot be reasonably detected at a level of about 10-20 copies per cell. In between these levels of positive and negative, a cell may be weakly positive for a particular marker he comparison between the expression level of a marker in a cell of the invention, and the expression level of the same marker in another cell, such as for example an embryonic stem cell, may preferably be conducted by comparing the two cell types that have been isolated from the same species. Preferably this species is a mammal, and more preferably this species is human. Such comparison may conveniently be conducted using a reverse transcriptase polymerase chain reaction (RT-PCR) experiment.

A population of stem cells is considered to express a marker if at least about 60% of the cells of the population show detectable expression of the marker. In other aspects, at least about 70%, at least about 80%, at least about 90% or at least about 95% or at least about 97% or at least about 98% or more of the cells of the population show detectable expression of the marker. In certain aspects, at least about 99% or 100% of the cells of the population show detectable expression of the markers. Expression may be detected through the use of an RT-PCR experiment, through fluorescence activated cell sorting (FACS), or through immunocytochemsitry using specific antibodies. It should be appreciated that this list is provided by way of example only, and is not intended to be limiting.

A population of stem cells is considered to be negative for a particular marker if at least about 60% of the cells of an isolated stem cell population does not show detectable expression of the marker. In other embodiments, at least about 70%, at least about 80%, at least about 90% or at least about 95% or at least about 97% or at least about 98% or at least about 99% or 100% of the cells of a population should not show any detectable expression of the marker. Again, lack of detectable expression may be proven through the use of an RT-PCR experiment, using FACS, or using immunocytochemistry.

The term "substantially pure", with respect to stem cells populations with enhanced enhanced clonogenic capacity and/or migratory capacity, refers to a population of stem cells that is at least about 75%, preferably at least about 85%, more preferably at least about 90%, and most preferably at least about 95% pure, with respect to stem cells making up a total cell population. Recast, the term "substantially pure" refers to a population of stem cells with enhanced clonogenic capacity and migratory capacity of the present invention that contain fewer than about 20%, more preferably fewer than about 10%, most preferably fewer than about 5%, of lineage committed cells in the original unamplified and isolated population prior to subsequent culturing and amplification.

COMPOSITION OF THE INVENTION

Another aspect of the invention relates to a composition, hereinafter composition of the invention, comprising: a) a cell co-culture comprising a stem cell, a CB-NK cell and IL-15, b) a stem cell of the invention, or c) a stem cell population of the invention.

Preferably, the NK cell of step a) is a CB NK cell.

In another preferred embodiment, the composition of the invention may comprise at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, of the stem cells of the invention, either calculated by number, or by weight or by volume of the composition.

In another preferred embodiment, the composition of the invention also comprises a pharmaceutically acceptable vehicle and/or a pharmaceutically acceptable excipient. In another preferred embodiment, the composition of the invention is a pharmaceutical composition.

As used herein, the term "active ingredient", "active substance", "pharmaceutically active substance", "active ingredient" or "pharmaceutically active ingredient" means any component which potentially provides a pharmacological activity or another different effect in diagnosing, curing, mitigating, treating, or preventing a disease, or which affects the structure or function of the human body or body of other animals. Examples of active ingredients of biological origin include growth factors, hormones, and cytokines. A variety of therapeutic agents is known in the art and may be identified by their effects. Certain therapeutic agents are capable of regulating cell proliferation and differentiation. Examples include chemotherapeutic nucleotides, drugs, hormones, non-specific (non-antibody) proteins, oligonucleotides (e.g., antisense oligonucleotides that bind to a target nucleic acid sequence (e.g., mRNA sequence)), peptides, and peptidomimetics. The pharmaceutical compositions of the present invention can be used in a treatment method in an isolated manner or together with other pharmaceutical compounds.

The term "pharmaceutically acceptable excipient" as used here refers to the fact that it must be approved by a regulatory agency of the federal government or a national government or one listed in the United States Pharmacopoeia or the European Pharmacopoeia, or some other pharmacopoeia generally recognized for use in animals and in humans. The term "vehicle" relates to a diluent, excipient, carrier or adjuvant with which the stem cells, progenitor cells or differentiated cells of the invention, the immortalized cells of the invention, as well as the cells of the cell population of the invention, must be administered; obviously, said vehicle must be compatible with the cells. Illustrative, non-limiting examples of said vehicle include any physiologically compatible vehicle, for example isotonic solutions (e.g. sterile saline solution (0.9% NaCI), phosphate -buffered saline solution (PBS), Ringer-lactate solution, etc.), optionally supplemented with serum, preferably with autologous serum; culture media (e.g. DMEM, RPMI, McCoy, etc.); or, preferably, a solid, semisolid, gelatinous or viscous support medium, such as collagen, collagen-glycosamine-glycan, fibrin, polyvinyl chloride, poly-amino acids, such as polylysine, or polyornithine, hydrogels, agarose, dextran sulphate silicone. Moreover, if desired, the support medium can, in special embodiments, contain growth factors or other agents. If the support is solid, semisolid, or gelatinous, the cells can be introduced in a liquid phase of the vehicle that is treated subsequently so that it is converted into a more solid phase. In some embodiments of the invention in which the vehicle has a solid structure, said vehicle can be configured according to the form of the lesion.

The pharmaceutical composition of the invention can, if desired, also contain, when necessary, additives for increasing and/or controlling the desired therapeutic effect of the cells, e.g. buffering agents, surface-active agents, preservatives, etc. The pharmaceutically acceptable carrier may comprise a cell culture medium which supports the cells' viability. The medium will generally be serum-free in order to avoid provoking an immune response in the recipient. The carrier will generally be buffered and/or pyrogen free. Also, for stabilizing the cellular suspension, it is possible to add chelating agents of metals. The stability of the cells in the liquid medium of the pharmaceutical composition of the invention can be improved by adding additional substances, such as, for example, aspartic acid, glutamic acid, etc. Said pharmaceutically acceptable substances that can be used in the pharmaceutical composition of the invention are generally known by a person skilled in the art and are normally used in the production of cellular compositions. Examples of suitable pharmaceutical vehicles are described in "Remington's Pharmaceutical Sciences" by E.W. Martin. Additional information on said vehicles can be found in any manual of pharmaceutical technology (that is, galenical pharmacy). The pharmaceutical composition of the invention will be administered in a suitable pharmaceutical form of administration. For this, the pharmaceutical composition of the invention will be formulated according to the chosen form of administration. The formulation will be adapted to the method of administration. In a special embodiment, the pharmaceutical composition is prepared in a liquid, solid or semisolid dosage form, e.g. in the form of suspension, in order to be administered by implanting, injection or infusion to the subject needing treatment. Illustrative, non- limiting examples include formulation of the pharmaceutical composition of the invention in a sterile suspension with a pharmaceutically acceptable excipient, e.g. an isotonic solution, for example, phosphate-buffered saline solution (PBS), or any other suitable, pharmaceutically acceptable vehicle, for administration to a subject parenterally, although other routes of administration can also be used.

The administration of the pharmaceutical composition of the invention to the subject who needs it will be carried out using conventional means. In a particular embodiment, said pharmaceutical composition of the invention can be administered to the subject parenterally using suitable devices such as syringes, catheters, trocars, cannulas, etc. In all cases, the pharmaceutical composition of the invention will be administered using equipment, apparatus and devices suitable for the administration of cellular compositions and known by a person skilled in the art. In another embodiment, direct administration of the pharmaceutical composition of the invention to the site that is intended to benefit may be advantageous. In this method, direct administration of the pharmaceutical composition of the invention to the desired organ or tissue can be achieved by direct administration (e.g. by injection, etc.) on the external surface of the affected organ or tissue by inserting a suitable device, e.g. a suitable cannula, by infusion (including reverse flow mechanisms) or by other means described in this patent or known in the art.

The variety of causes influencing cell homing is quite high, then the capability of CB-NK cells to increase it will decrease variability ensuring therapeutic efficacy in many cell therapy treatments. The efficacy of a pharmacological drug is dependent on the via by which is being administered. For example, in order to enhance efficacy and availability, the method of administration of MSCs should hence facilitate homing of MSCs to the desired tissue. Intravenous infusion is one of the major routes of administration of MSC. When MSCs are infused systemically, they are trapped into capillary beds of various tissues, especially the lungs which act as a filter

Therefore, intra-arterial injection of MSCs has been assessed in humans (Ruiz-Salmeron et al., 201 1 . Cell Transplantation 20(19): 1629-1639). But even in the late stage of the peripheral artery disease (e.g. in the critical ischaemia of the limbs) intrarterially injected cells enter into the systemic circulation and induce changes in distal tissues. Delivery of MSCs subacute spinal cord injury (SCI), via the vertebralis artery leads to a greater functional improvement than when cells were administered via the intravenous route. However, delivery of cells in an artery may lead to "microvascular occlusions" , specially when autologous cells are derived form inflamed tissues. It has been shown that to treat myocardial infraction (Ml), direct delivery of bone marrow cells or MSCs in the heart or close to the site of injury enhances the number of cells found in the peri-infarct region.

The pharmaceutical composition of the invention can be stored until the moment of its application by the conventional methods known by a person skilled in the art. For short-term storage (less than 6 hours), the pharmaceutical composition of the invention can be stored at or below room temperature in a sealed container, supplemented or not with a nutrient solution. Medium-term storage (less than 48 hours) is preferably carried out at 2-8[deg.]C, and the pharmaceutical composition of the invention includes, in addition, an iso-osmotic, buffered solution in a container made of or lined with a material that prevents cellular adhesion. Longer- term storage is preferably carried out by means of suitable cryopreservation and storage in conditions that promote the retention of cellular function.

In a concrete embodiment, the pharmaceutical composition of the invention can be used in combination therapy. Said additional medicinal products can form part of the same pharmaceutical composition or can, alternatively, be supplied in the form of a separate composition for simultaneous or successive (sequential in time) administration relative to the administration of the pharmaceutical composition of the invention.

KIT OF PARTS OF THE INVENTION

Another aspect of the invention relates to a kit of parts, hereinafter kit of parts of the invention, comprising:

a) a stem cell, a NK cell and IL-15, or

b) a stem cell and an IL-15 activated NK cell. Preferably, the NK cell of the steps a) or b) is a CB NK cell.

The kit of parts may comprise separate formulations of cells, preferably stem cells, and NK cells, preferably activated NK cells, and more preferably activated CB NK cells. The separate formulations may be administered sequentially, separately and/or simultaneously (optionally repeatedly). Thus, the active ingredients can be administered either as a part of the same pharmaceutical composition or in separate pharmaceutical compositions. Stem cells can be administered prior to, at the same time as, or subsequent to administration of activated NK cells, or in some combination thereof.

MEDICAL USES OF THE INVENTION

An infectious, inflammatory, genetic or degenerative disease, physical or chemical damage, or blood flow interruption, can cause cell loss from a tissue or organ. This cell loss would lead to an alteration of the normal function of said tissue or organ; and consequently lead to the development of diseases or physical consequences reducing the person's quality of life. Therefore, attempting to regenerate or and reestablish the normal function of said tissues or organs is important. Cell recruitment of resident progenitors to the damaged tissue, such as heart, brain or ischaemic legs is a major challenge to improve any cell based therapy. The process requires homing and engraftment of stem or progenitor cells. Major strategies to improve stem or progenitor cell homing used so far included the use granulocyte colony-stimulating factor (G- CSF), parathyroid hormone, statins, erythropoietin, and others. All these strategies are based on an improvement of stem or progenitor cell mobilization from the bone marrow. Strategies that have been shown to be successful are no major impact on hemodynamics was found in most of them. Then, improvement of stem cell homing is a major challenge in the development of successful cell based therapies but not yet improved to a clinical relevant status. The authors of the present invention showed that the expression of homing receptors by CBSC was modified in the presence of aNK but not rNK cells. Of particular interest was the higher expression of CXCR4 observed by CBSC in the presence of aNK cells.

Also, they performed transwell migration assays to analyze the effect of aNK cells on the migration capacity of CBSC, alone and in the presence of CB-NK cells, using SDF1 a as a stimulus for migration. Surprisingly they found that CB-NK cells play an important role in stem cells migration and engraftment, especially in umbilical cord blood stem cells (CBSC). CBSC have many attractive properties such as low incidence and severity of graftversus-host disease (GvHD), a strong graft-versus-leukemia (GvL) and an increased tolerance to HLA mismatch are highly relevant. As is shown in the examples of the invention, the activated CB-NK cells also promote homing of MSC on the bone marrow stroma promoting not only stem cell engraftment but bone formation, osteoblast activity and trabecular borne volume in osteoporotic bones.

Then, in another aspect relates to the stem cell of the invention, the stem cell population of the invention, the composition of the invention, or the kit of parts of the invention, for its use in therapy or for its use as a medicament.

Direct contact between CBSC and activated NK cells is required to enhance CBSC clonogenic capacity. The integrins beta-7, CD1 1 a, CD49d and CD49e play key roles in HSC homing and maintenance within the BM niche. Whilst it is unknown whether these integrins are important for the interaction between CBSC and CB-NK cells, the inventors observed regulation of the expression of these molecules by CBSC after co-culture with aNK cells. Therefore, they assessed whether blocking these integrins using antibodies would influence the effect of aNK cells on CBSC clonogenic capacity. Blocking beta-7 integrin, CD1 1 a, CD49d or CD49e alone or in combination significantly reduced the ability of CB aNK cells to enhance CBSC clonogenic capacity (Figure 8), suggesting that direct contact between CBSC and CB-NK cells via these specific integrins is required.

Activated NK cells induce CXCL-9 secretion by CBSC and increase their clonogenicity In order to identify molecular pathways that are modified in CBSC after culture with rNK cells or aNK cells, the inventors performed gene expression analysis by microarray, comparing CBSC co- culturedwith rNK cells or aNK cells. Clear transcriptional changes were observed when CBSC were cultured with NK cells. In particular, the expression of 489 genes was significantly changed at least 1.5 fold (p < 0.05) in CBSC after co-culture with rNK cells while the expression of 1970 genes was significantly modified when CBSC were cultured with aNK cells (Figure 9 A- B). Notably, the inventors found that one gene, CXCL-9, was particularly upregulated by CBSC after culture with aNK cells (274 fold-change (FC)). The inventors confirmed that CBSC secreted high levels of CXCL-9 after co-culture with aNK cells but not with rNK cells (Figure 9C), suggesting that this chemokine might play a key role in regulating CBSC functions. We then assessed the effect of CXCL9 on CBSC functions by treating CBSC with different doses of recombinant CXCL9 followed by CFU assays. Treating CBSC with 10, 50 or 100 ng/ml of CXCL9 significantly increased CBSC clonogenicity (p < 0.05) while higher CXCL9 concentrations had no effect on CBSC function (Figure 9D). Another aspect relates to the stem cell of the invention, the stem cell population of the invention, the composition of the invention, or the kit of parts of the invention, for its use to partially or completely increase, restore or replace the functional activity of a diseased or damaged tissue or organ. Hematopietic Cord blood transplantation (CBT) offers many advantages including rapid accessibility of the graft, less stringent HLA matching, a lower incidence of Graft versus Host Disease (GvHD) and a preserved Graft versus Leukemia (GvL) effect as compared to transplantation performed with bone marrow (BM) or mobilized peripheral blood (mPB) hematopoietic stem cells (HSC) Graft-versus-host disease in children who have received a cord-blood or bone marrow transplant from an HLA-identical sibling. However, some of the main limitations of CBT are a lower number of nucleated cells per cord blood (CB) unit, delayed engraftment and immune reconstitution and a higher incidence of infection. It has been described that the low HSC dose, the leukocyte content of the graft and ineffective CB CD34+ cells homing to the BM are factors associated with graft failure post-CBT. CD34+ cells (HSC) must migrate to the BM in order to engraft and facilitate long-term immune reconstitution.

The activated NK cell, preferably cord blood NK cells, enhance the clonogenic capacity of hematopoietic CD34+ cells. The authors of the present invention describe the effect of accessory cells on CD34+ cells engraftment in NSG mice and identified CB NK cells as a key population that influences CD34+ cells engraftment in vivo. Co-culture of CB NK cells with CD34+ cells increased CXCR4 expression on CD34+ cells, which translated into enhanced chemotaxis towards SDF-1 ain vitro. CB NK cells also enhanced CD34+ cells clonogenic capacity as assessed by short-term and long-term in vitro cultures, potentially by inducing CXCL9 secretion by CD34+ cells. The effect on clonogenic capacity was contact dependent as blocking of key integrins expressed by CD34+ cells prevented the effect of CB NK cells. These data demonstrate a novel effect of CBNK cells on CD34+ cells that could be utilized to improve the outcome of Cord BT.

Then, another aspect relates to the stem cell of the invention, the stem cell population of the invention, the composition of the invention, or the kit of parts of the invention, for its use in hematopoietic stem cell transplantation (HSCT).

Hematopoietic stem cell transplantation (HSCT) is the transplantation of multipotent hematopoietic stem cells, usually derived from bone marrow, peripheral blood, or umbilical cord blood. It may be autologous (the patient's own stem cells are used) or allogeneic (the stem cells come from a donor). In a preferred embodiment, the hematopoietic stem cell transplantation is an umbilical cord blood transplantation.

The examples of the invention show that CB-NK cells enhance CD34+ cells engraftment in NSG mice. The inventors first analyzed the effects of CB-NK cells, CD4+ T cells and CD8+ T cells isolated from the same CB unit on CD34+ cells engraftment in NSG mice. They chose to treat CB-NK cells with IL-15 for 4h as this led to significant NK cell activation as shown by upregulation of the activation marker CD69(p < 0.0001 ) (Figure 4A),without altering their cytolytic function (data not shown). They found that mice transplanted with CBSC and aNK cells showed higher levels of hCD45+ cell engraftment in the BM compared to mice that received any of the other cell combinations (Figure 4B), identifying CB-NK cells as a key population that influences CBSC engraftment in vivo.

Hematopoietic stem cell transplantation often performed for patients with certain cancers of the blood or bone marrow, such as multiple myeloma or leukemia. In these cases, the recipient's immune system is usually destroyed with radiation or chemotherapy before the transplantation. Infection and graft-versus-host disease are major complications of allogenic HSCT.

Moreover, another aspect relates to the stem cell of the invention, the stem cell population of the invention, the composition of the invention, or the kit of parts of the invention, for its use in the treatment, amelioration or prevention of a bone marrow disease or disorder.

In a preferred embodiment the bone marrow disease or disorder is selected from the list consisting of: a leukemia, a lymphoma, a myeloproliferative disease, a myelodysplasia syndrome and a plasma cell disorder, or combinations thereof.

In another preferred embodiment, the leukemia is selected from the group consisting of Acute Lymphocytic Leukemia (ALL), Acute Myelocytic Leukemia (AML), Chronic Lymphocytic Leukemia (CLL), Chronic Myelocytic Leukemia (CML), Chronic Myelomonocytic Leukemia (CMML), stem cell leukemia, or combinations thereof.

In another preferred embodiment the lymphoma is selected from the group consisting of Anaplastic Large-Cell Lymphoma (ALCL), Hodgkin's lymphoma, non-Hodgkin's lymphoma, or combinations thereof. In another preferred embodiment the myelodysplastic syndrome is selected from the group consisting of Refractory anemia (RA), Refractory anemia with ringed sideroblasts (RARS), Refractory cytopenia with multilineage dysplasia (RCMD), Refractory cytopenia with multilineage dysplasia and ringed sideroblasts (RCMD-RS), Refractory anemia with excess blasts I and II; 5q- syndrome, myelodysplasia unclassifiable, or combinations thereof.

In another preferred embodiment the myeloproliferative disorder is selected from Polycythemia Vera (PV), Essential Thrombocythemia (ET), Chronic Idiopathic Myelofibrosis (MF), or combinations thereof.

In another preferred embodiment the plasma cell disease is a plasma cell neoplasm selected from the group consisting of multiple myeloma, plasmacytoma, macroglobulinemia, monoclonal gammopathy of undetermined significance (MGUS), or combinations thereof. In another preferred embodiment the bone marrow disease or disorder is a hemoglobinopathy selected from the group consisting of sickle-cell disease and S a- thalassemia.

Another aspect of the present invention relates to the stem cell of the invention, the stem cell population of the invention, the composition of the invention, or the kit of parts of the invention, for its use in the treatment, amelioration or prevention of ischemia or peripheral artery disease.

In a preferred embodiment the ischemia or peripheral artery disease is selected from the list consisting on: cerebrovascular ischemia, renal ischemia, pulmonary ischemia, limb ischemia, ischemic cardiomyopathy and myocardial ischemia or combinations thereof. In another preferred embodiment the ischemia or peripheral artery disease is the critical limb ischemia.

Another aspect of the invention relates to the stem cell of the invention, the stem cell population of the invention, the composition of the invention, or the kit of parts of the invention, for its use in the treatment, amelioration or prevention of myocardial infarction.

Another aspect of the invention relates to the stem cell of the invention, the stem cell population of the invention, the composition of the invention, or the kit of parts of the invention, for its use in the treatment, amelioration or prevention of stroke. Another aspect of the invention relates to the stem cell of the invention, the stem cell population of the invention, the composition of the invention, or the kit of parts of the invention, for its use in the treatment, amelioration or prevention of erectile dysfunction. Another aspect of the invention relates to the stem cell of the invention, the stem cell population of the invention, the composition of the invention, or the kit of parts of the invention, for its use in the treatment, amelioration or prevention of wound healing. In a preferred embodiment, relates to the stem cell of the invention, the stem cell population of the invention, the composition of the invention, or the kit of parts of the invention, for its use to promote wound healing in Crohn's fistula, diabetic ulcers or radiotherapy caused ulcers and fistulae. Another aspect of the invention relates to the stem cell of the invention, the stem cell population of the invention, the composition of the invention, or the kit of parts of the invention, for its use to enhance hematopoietic CD34+ stem cell engraftment.

Another aspect of the invention relates to the stem cell of the invention, the stem cell population of the invention, the composition of the invention, or the kit of parts of the invention, for its use in the treatment, amelioration or prevention of diabetic neuropathy.

Another aspect of the invention relates to a method of enhancing stem cell homing and engraftment potential comprising the co-culture of said stem cell with at least a NK cell as described before.

The effect of CB-NK cells on the short-term and long-term clonogenic capacity of CBSC was assessed using CFU assays and cobblestone cultures followed by long-term culture (LTC-IC) respectively. CBSC were cultured for 4 h in the presence of rNK cells or aNK cells prior to assessing their clonogenic capacity. A higher number of CFUs was obtained when CBSC were co-cultured with aNK cells (P < 0.01 )( Figure 7B). Although the number of CFUs obtained from CBSC cultured with rNK cells was higher than the CBSC alone control this difference did not reach significance (Figure 7A). Co-culture of CBSC with aNK cells, but not with rNK cells, also increased the number of CFUs obtained from long-term LTC-IC cultures (P < 0.05) (Figure 7C and D, respectively), suggesting that aNK cells enhance both the short and long-term clonogenic capacity of CBSC. Finally, the inventors assessed whether NK cells could impact on the cell cycle of CBSC. However, we found that rNK or aNK cells had no effect on CBSC cell cycle (Figure 7E). Then, another aspect of the invention refers to the use of activated NK cells to enhance the short-term and long-term clonogenic capacity of cells, preferably stem cells, more preferably CD34+ cells, still more preferably hematopoietic CD34+ cells and even still more preferably CBSC. In a preferred embodiment, the cell is selected from stem cells, progenitor cells, neutrophils, macrophages and T-cells. In another preferred embodiment the stem or progenitor cells may be embryonic stem cells, adult stem cells, expanded stem cells, placental stem cells, bone marrow stem cells, amniotic fluid stem cells, neuronal stem cells, cardiomyocyte stem cells, placental stem cells, endothelial progenitor cells, circulating and mobilized peripheral blood stem cells, muscle stem cells, germinal stem cells, adipose tissue derived stem cells, exfoliated teeth derived stem cells, hair follicle stem cells, dermal stem cells, parthenogenically derived stem cells, reprogrammed stem cells such as induced pluripotent stem cells, somatic nuclear transfer cells, or combinations thereof.

In another preferred embodiment, the T-cell is selected from the list consisting on T cells, activated T-cells, helper T cells, cytolytic T-cells, memory T-cells, regulatory T-cells or reprogrammed cells, or a combination thereof that may from a heterogeneous population of T- cells.

To get a better understanding of the effects of rNK cells or aNK cells on CBSC, the inventors performed a microarray analysis, comparing rNK cells to aNK cells. The expression of 148 genes was significantly upregulated at least 1 .5 fold (p < 0.05), including IFN-γ and TNF-β, and 230 were significantly down regulated at least 1.5 fold (p < 0.05), including CXCR3 and CXCR4, in aNK cells compared to rNK cells (Figure 10A and Table S2). Notably, we confirmed that following stimulation with IL-15 aNK cells secrete significantly more IFN-γ and TNF-β than rNK cells (Figure 10B).

Then, another aspect of the invention refers to activated NK cells for use in the prevention, treatment or amelioration of IFN-γ mediated diseases. In a preferred embodiment of this aspect of the invention, the IFN-γ mediated disease is an immune-mediated inflammatory disease, an autoimmune diseases, or an inflammatory disorder.

In another preferred embodiment, the IFN-γ mediated disease is selected from the group consisting of: lupus erythematosus, scleroderma, sclerodermoid disorders, vasculitis syndromes, occlusive vasculopathies, autoinflammatory syndromes, syndromes from innate immunity dysregulation (eghidradeitissuppurativa, pustular psoriasis), neutrophilicdermatoses, psoriasis, cardio-metabolic risk of chronic inflammation, atopic dermatitis, chronic itch, febrile dermatoses, psoriatic arthrithis, autoimmune bollous diseases, eosinophilicdermatoses, atopic eczema, urticaria, Bechet's disease, neutrophilicdermatoses, hidradenitissuppurativa, pustular psoriasis, autoimmune bullous diseases, chronic hepatitis B virus infection, sepsis induced by Streptococcus pneumoniae or Escherichia coli, diabetes induced by coxsackie virus B4, arthritis, sarcoidosis, collagenoses, rheumatism, hemolytic anemia, immune form of idiopathic thrombocytopenic purpura, eczemas, nephritis, myasthenia gravis, Hashimoto disease, autoimmune diseases of the organs of sight and hearing, multiple

sclerosis, Meniere's disease, Parkinson's disease, pemphigus, schizophrenia, Crohn's disease or any combinations thereof.

In another preferred embodiment, the IFN-γ mediated disease is allograft rejection. In another preferred embodiment, the IFN-γ mediated disease is graft versus host disease. In another more preferred embodiment, the graft versus host disease is the cutaneous graft versus host disease. In another preferred embodiment, the IFN-γ mediated disease is a skin inflammatory disease. More preferably, the skin inflammatory disease is selected from the list consisting of: psoriasis, allergic contact dermatitis, atopic dermatitis, cutaneous graft versus host disease, cutaneous cell lymphoma, metal allergy, lichen planus, or any combinations thereof. In the examples of the invention, a higher number of CFU was observed for CBSC in the presence of aNK cells from either short-term or long-term cultures suggesting that aNK cells not only modify CBSC trafficking capacity but also their clonogenicity. Also, experiments show that activation of CB-NK cells increase the expression of homing receptor CXCR4 in stem cells and enhancing migration and engraftment.

Finally, the inventors showed by gene expression profiling analysis and ELISA that CBSC secreted high levels of CXCL9 after co-culture with aNK cells. Moreover, the microarray analysis also revealed that CBSC expressed higher messenger levels of genes that are regulated by IFN-γ and that aNK cells secreted significantly more IFN-γ and TNF-β than rNK cells. It could be that the CB-NK cells secrete IFN-γ, which in turn induced CXCL9 expression by CBSC, acting on their clonogenicity as the inventors showed that recombinant CXCL9 increased CBSC clonogenicity.

Along the description and claims, the word "comprises" and variants thereof do not intend to exclude other technical features, supplements, components or steps. For persons skilled in the art, other objects, advantages and features of the invention will be understood in part from the description and in part from the practice of the invention. The following examples and drawings are provided by way of illustration and they are not meant to limit the present invention. EXAMPLES OF THE INVENTION

The following specific examples provided in this patent document serve to illustrate the nature of the present invention. These examples are included only for illustrative purposes and must not be interpreted as limiting to the invention which is claimed herein. Therefore, the examples described below illustrate the invention without limiting the field of application thereof.

Cord blood samples and cell purification Fresh CB samples were obtained with written informed consent from the Anthony Nolan Cord Blood Bank, Nottingham, UK (National Research Ethics Committee reference 10/H0405/27) or from Dr Alicia Esparza Clinic (MEDIMAR, Alicante) and processed within 24 h of collection. CB (cord blood) mononuclear cells (CBMCs) were isolated by density gradient centrifugation using Ficoll-Paque PLUS (GE Healthcare). CBSC were isolated using the CD34 microbead kit (Miltenyi Biotec) (Jaatinen T, Laine J. Isolation of hematopoietic stem cells from human cord blood. Curr Protoc Stem Cell Biol 2007 Jun; Chapter 2: Unit 2A 2) to a purity of 98.4 % ± 0.75. CB NK cells were isolated using the NK cell isolation kit (Miltenyi Biotec), to a purity of 90.39% ± 3.35. T cells were labeled with PE-conjugated CD4 or CD8 antibodies respectively and isolated from CB usinganti-PE MultiSort MicroBeads (Miltenyi Biotec) with purities of 90.16% ± 0.76 and 81.66% ± 1 1.06 respectively.

Flow cytometry

Cells were stained with fluorophore-conjugated antibodies at 4°C for 10 min (or for 45 min for anti-CXCR4 and anti-CXCR7 antibodies), washed and re-suspended in 1 X PBS containing 10% FBS. A FACSCalibur flow cytometer (Becton Dickinson) or a LSRFortessa flow cytometer (Becton Dickinson) were used to acquire data and FlowJo software (TreeStar) was used for data analysis. The following monoclonal antibodies were purchased from BD Biosciences: CD3 (SK7), CD4 (SK3), CD8 (SK1 ), CD1 1 a (HI 1 1 1 ), CD29 (TS2/16), CD34 (581 ), CD44 (Bu52), CD45 (HI30), CD49d (9F10), CD49e (IIA1 ), CD49f (GoH3), CD56 (B159), CD69 (L78), CD133 (293C3), CD162 (KPL-1 ), CXCR4 (12G5), CXCR7 (358426), NKp44 (P44-8) and β7 integrin (12G5). Cell viability was assessed using Annexin Vand 7AAD (BD Biosciences). For cell cycle analysis, cells were fixed with 70 % Ethanol/30 % PBS for at least 1 h at 4° C. The fixed cell pellet was then incubated for 10 min at RT with RNAse at 0.17 mg/mL. To stain the DNA, the cells were incubated for 1 h at 37 °C with propidium iodide at 36 ug/mL and then analyzed by flow cytometry.

Cell culture

MS-5 cells were a kind gift of Dr Bonnet (CRUK, London). Cells were cultured in alpha-MEM with 10 % FBS, 2 mM L-glutamine and 2 mM sodium pyruvate. CBNK cells were activated with 20 ng/mL IL-15 (Peprotech) in RPMI-1640 containing 10 % FBSand 50 μΜ β-MEfor 4 h.CBSC were cultured either alone or with resting NK (rNK) cells or activated NK (aNK) cells for 4 h at a ratio of 1 to 5 CBSC to CB NK cells.

Murine Hindlimb ischaemia animal experiments

Experiments were approved by the Institutional Animal Experimental Committee that conforms to the Guide for the Care and use of Laboratory Animals published by the US National Institutes of Health (NIH Publication, 8th edition, revised 201 1 ). Mice were anaesthetized by intraperitoneal (i.p.) injection of dexmedetomidine (1 .0 mg/kg, Dexdomitor, Zoetis) and ketamine (70.0 mg/kg, Alfasan). Unilateral double ligation of the right femoral artery was performed and a sham operation, in which the femoral artery was dissected, was performed on the left side. Adequacy of the anaesthesia and surgical tolerance was monitored by close observation and reflex response tests. Animal were allowed to recover for 3 days prior to initiation of treatments. The treatment group received both intraarterial and/or intramuscular injections of adipose- derived mesenchyal stem cells (1 x 10 /kg of body weight) alone or in combination with CB-NK cells. Animals were monitored during a 4-week period.

Laser Doppler perfusion imaging Hindlimb perfusion was measured using LDPI (Moorl_DI2, Moor Instruments, Devon, UK). LDPI was performed under anaesthesia directly before and after femoral artery ligation, as well as at 5, 7, 14, and 28 days following ligation. Excess hair from the hindlimb was removed using depilatory cream and mice were placed on a heating plate to minimize temperature variation. Dedicated software was used to quantify tissue perfusion. To account for variables such as ambient light, temperature, and experimental procedures, perfusion was calculated in the foot and expressed as a ratio of right (ischaemic) to left (non-ischaemic) hindlimb.

Cell migration and angiogenesis assay: Endothelial progenitor cells (2 x 104) were isolated, resuspended in 250 μΙ of endothelial cell basal medium and seeded in the upper chamber of a Costar Transwell chamber (6.5 mm, 5 μηη pore size, Coring NY). The chamber was placed n a24 well culture dish containing 500 μΙ of endothelial basal medium under different experimental conditions. After 24 h at 37°C transmigrated cells were counted. The angiogenesis assay was performed as described by the manufacturers (Cell Systems, Clonetics)

Osteoporosis Animal models for postmenopausal osteoporosis are generated by ovariectomy. Bone loss occurs in estrogen deficiency due to enhanced bone resorption and impaired osteoblast function. Estrogen receptor a induces osteoclast apoptosis, but the mechanism for impaired osteoblast function remains to be clarified.

Bone mass is determined by the balance between the activities of osteoblasts, which form bone, and those of osteoclasts, which resorb bone. In mice, the loss of cancellous bone but not cortical bone occurs soon after ovariectomy, and estrogen replacement with 17-3-estradiol in ovariectomized mice prevents bone loss (R.L. Jilka, G. Hangoc, G. Girasole, G. Passeri, D.C. Williams, J.S. Abrams, B. Boyce, H. Broxmeyer, S.C. Manolagas (1992) Increased osteoclast development after estrogen loss: mediation by interleukin-6. Science, 257 (1992), pp. 88-91 ). In contrast with the intra-medullar-infused mesenchymal stromal cells that may be detected in epiphysis or diaphysis of the distal femurs and/or proximal tibiae, infused MSC were much less effective (intra-venous-infused MSC were not detected in any location).

Colony forming unit assays

CBSC were cultured with rNK cells or aNK cells or with CXCL9 (Peprotech) at different concentrations for 4 hrs. A minimum of 200 CBSC were plated in MethoCult GF H-84434 (Stemcell Technologies) and cultured for 14 days at 37 °C, 5% C02. Colony formation was enumerated using an inverted microscope at the end of culture. For integrin blocking experiments, CBSC were incubated with blocking antibodies (eBioscences) against β7 integrin (FIB504), CD1 1 a (HI1 1 1 ), CD49d (9F10) and CD49e (IIA1 ) at 10 g/mL for 1 h and then washed before co-culture with NK cells and CFU assays.

LTC-IC assays

CBSC (500 per well) were seeded on a feeder layer of irradiated MS-5 cells (30 Gray) in alpha- MEM, 20 % FBS, 10-5 M hydrocortisone, 50 μΜ β-ΜΕ, at ten replicates per condition, for either CBSC alone or co-cultured with rNK or aNK cells for 4 h. Wells were scored for cobblestone areas after 4 weeks and adherent cells were used to perform CFU assays as described.

Migration assays Transwell migration assays were performed using 5μηι polycarbonate membrane HTS 96-well transwell plates (Corning) coated with fibronectin (20 μg mL). CBSC were plated in the upper chamber of the transwell plate either alone or in the presence of rNK or aNK cells. Migration to the lower chamber was assessed by CFU assay after 4 husing a standard SDF-1 a concentration of 125 ng/mL (Peled A, Petit I, Kollet O, Magid M, Ponomaryov T, Byk T, et al. Dependence of human stem cell engraftment and repopulation of NOD/SCID mice on CXCR4. Science 1999 Feb 5; 283(5403): 845-848after 3 h using a sub-optimal concentration of 50 ng/mL; JA. C. Adhesion, migration, and homing of murine hematopoietic stem cells and progenitors. Methods Mol Biol 201 1 ; 750: 187-196). The chemotactic index was calculated as the ratio of the number of cells that migrated towards SDF-1 a to the number of cells that migrated towards medium only.

Microarray analysis

CBSC and rNK or aNK cells were co-cultured for 4 h and then stained with anti-CD34-PE (BD Biosciences) for re-isolation of CBSC by sorting using a FACSAria cell sorter (BD Biosciences). RNA was isolated from sorted CBSC, rNK or aNK cells using the RNeasy Micro kit (Qiagen). Samples were processed using GeneChip Whole Transcript Sense Target Labeling assays using the Ambion WT Expression kit and Affymetrix GeneChip WT Terminal Labeling and Controls kit (Affymetrix). The resulting ssDNAs were hybridized to the GeneChip human Gene 2.0 ST Array (Affymetrix) and microarray analysis was performed by the UCL Genomics Facility, Institute of Child Health (London, UK). Image reads were processed using Affymetrix software and background was corrected and normalized using the RMA algorithm with GeneSpring software (Agilent Technologies). Differentially expressed genes were analyzed using GeneSpring software. Data are available from the EMBL-EBI/ArrayExpress repository under accession E-MTAB-2531 for the CBSC analysis and E-MTAB-2847 for the NK cell analysis.

ELISA

Human CXCL9 was quantified in culture supernatants from co-cultures between CBSC and NK cells using the Human MIG Instant ELISA (eBioscience). No CXCL9 secretion was detected from cultures of NK cells alone. Human IFN-γ and TNF-β secretion following CB-NK cell activation were measured using the corresponding Human Instant ELISA (eBioscience).

CBSC engraftment in NSG mice

NOD/SCID IL-2 Rynull (NSG) mice (males and females, 8 to 10 weeks old) were irradiated with 3.75 Gray. Irradiated NSG mice were injected intravenously 24 h later with 20,000 CD34+CBSC alone or with accessory cells from the same CB unit at a ratio of 5 to 1 accessory cells to CBSC. After 10 weeks, the level of CBSC engraftment in the BM was assessed for each mouse by analysis of human CD45 expression using flow cytometry. Animal experiments were performed according to the UK Home Office Regulations under the project license 80/2456. For each experiment, 4-6 mice were used per group. Mice were randomly assigned to each group.

Statistical analysis

Statistical analysis was performed using Prism 5 (GraphPad Software Inc., USA). Data are presented as means and SD. Paired or unpaired t tests were used to analyze results obtained from in vitro and in vivo experiments respectively. P values less than 0.05 were considered statistically significant.

RESULTS

We first analyzed the effects of CB-NK cells, CD4+ T cells and CD8+ T cells isolated from the same CB unit on CBSC engraftment in NSG mice. For this study, resting cells were used except for where we compared resting (resting CB-NK) or activated (activated CB-NK) NK cells. Moreover, we have identified that higher concentration of IL-2 was required to activate CB-NK cells as compared to PB NK cells and that IL-15 led to a better activation of CB-NK cells as compared to IL-2. We chose to treat CB-NK cells with IL-15 for 4 h at a concentration which led to significant NK cell activation as shown by up-regulation of the activation marker CD69 (p < 0.0001 ) (Figure 4A), without altering their cytolytic function or expression of activating or inhibitory receptors. We found that mice transplanted with CBSC and activated CB-NK cells showed higher levels of hCD45+ cell engraftment in the BM compared to mice that received any of the other cell combinations (Figure 4B), identifying CB-NK cells as a key population that influences CBSC engraftment in vivo.

We therefore chose to focus on the effects of CB-NK cells on CBSC thereafter. However, upon further analysis of the effects of NK cells on CBSC in vivo, we found that CBSC constantly engrafted better in the presence of CB-NK cells, either resting CB-NK cells (p < 0.05) or activated CB-NK cells (p < 0.05), when compared to NSG mice infused with CBSC only (Figure 4C). In order to assess the effect of CB NK cells on CBSC, taking into account the variability between research grade CB units, as well as the natural variability in NK cell activation state, the same experiment was performed using six different CB units and co-culturing CBSC with resting CB-NK cells or activated CB-NK cells for 4 h prior to infusion into NSG mice. Variable levels of engraftment were obtained from each CB unit; however, higher levels of engraftment were systematically observed in mice that received CBSC plus either resting CB-NK cells or activated CB-NK cells with all the CB units used (Figure 4D). Notably, we didn't observe any correlation between levels of engraftment and levels of CD69 on resting CB-NK or activated CB-NK cells.

We next assessed whether CB-NK cells affect the homing properties of CBSC by analyzing the expression of key adhesion molecules and chemokine receptors for homing to the BM. Receptor expression by CBSC was assessed by flow cytometry following 4 h co-culture with resting CB-NK or activated CB-NK cells. Importantly, co-culture had no effect on CBSC or CB- NK cell viability. We found that CBSC expressed significantly lower levels of CD1 1 a (p < 0.05), CD29 (p < 0.01 ), CD44 (p < 0.05) and integrin beta-7 (p < 0.05) after co-culture with activated CB-NK cells but not with resting CB-NK cells (Figure 5A). Interestingly, we observed significantly higher expression levels of the chemokine receptors CXCR4 (p < 0.001 ) and CXCR7 (p < 0.05) on CBSC co-cultured with aNK cells compared to CBSC alone or co-cultured with rNK cells (Figure 5B).

As an increased expression of CXCR4 and CXCR7 by CBSC was observed after co-culture with activated CB-NK cells, we studied whether this led to an increased response of CBSC to SDF-1 a in vitro. Migration of CBSC in vitro towards SDF-1 a was significantly enhanced in the presence of activated CB-NK cells when compared to CBSC alone (P < 0.05) (Figure 6A). Though an increased chemotactic index was observed in the presence of resting B-NK cells, it was not significant when compared to CBSC alone. We then performed transwell migration assays using a sub-optimal dose of SDF-1 a and a shorter incubation time. In line with the results obtained using a standard concentration of SDF-1 a, activated CB-NK cells and not resting CB-NK cells specifically enhanced the migration capacity of CBSC towards a sub- optimal dose of SDF-1 a (P < 0.05) (Figure 6B).

The effect of CBNK cells on the short-term and long-term clonogenic capacity of CBSC was assessed using CFU assays and cobblestone cultures followed by long-term culture (LTC-IC) respectively. CBSC were cultured for 4 h in the presence of resting CB-NK cells or activated CB-NK cells prior to assessing their clonogenic capacity. A higher number of CFUs was obtained when CBSC were co-cultured with activated CB-NK cells (P < 0.01 ) (Figure 7B). Although the number of CFUs obtained from CBSC cultured with resting CB-NK cells was higher than the CBSC alone control this difference did not reach significance (Figure 7A). Co- culture of CBSC with activated CB-NK cells, but not with resting CB-NK cells, also increased the number of CFUs obtained from long-term LTC-IC cultures (P < 0.05) (Figure 7C and D respectively), suggesting that activated CB-NK cells enhance both the short and long-term clonogenic capacity of CBSC. Finally, we assessed whether NK cells could impact on the cell cycle of CBSC. However, we found that resting CB-NK or activated CB-NK cells had no effect on CBSC cell cycle (Figure 7E).

The integrins beta-7, CD1 1 a, CD49d and CD49e play key roles in HSC homing and maintenance within the BM niche. Whilst it is unknown whether these integrins are important for the interaction between CBSC and NK cells, we observed regulation of the expression of these molecules by CBSC after co-culture with activated CB-NK cells. Therefore, we assessed whether blocking these integrins using antibodies would influence the effect of activated CB-NK cells on CBSC clonogenic capacity. Blocking beta-7 integrin, CD1 1 a, CD49d or CD49e alone or in combination significantly reduced the ability of CB activated CB-NK cells to enhance CBSC clonogenic capacity (Figure 8), suggesting that direct contact between CBSC and CB-NK cells via these specific integrins is required. In order to identify molecular pathways that are modified in CBSC after culture with resting CB- NK cells or activated CB-NK cells, we performed gene expression analysis by microarray comparing CBSC co-cultured with resting CB-NK cells or activated CB-NK cells. Clear transcriptional changes were observed when CBSC were cultured with CB-NK cells. In particular, the expression of 489 genes was significantly changed at least 1 .5 fold (p < 0.05) in CBSC after co-culture with resting CB-NK cells while the expression of 1970 genes was significantly modified when CBSC were cultured with activated CB-NK cells (Figure 9A-B). Notably, we found that one gene, CXCL-9, was particularly up-regulated by CBSC after culture with activated CB-NK cells (274 fold-change (FC)). We confirmed that CBSC secreted high levels of CXCL-9 after co-culture with activated CB-NK cells but not with resting CB-NK cells (Figure 9C), suggesting that this chemokine might play a key role in regulating CBSC functions. We then assessed the effect of CXCL9 on CBSC functions by treating CBSC with different doses of recombinant CXCL9 followed by CFU assays. Treating CBSC with 10, 50 or 100 ng/ml of CXCL9 significantly increased CBSC clonogenicity (p < 0.05) while higher CXCL9 concentrations had no effect on CBSC function (Figure 9D). To get a better understanding of the effects of resting CB-NK cells or activated CB-NK cells on CBSC, we performed a microarray analysis, comparing resting CB-NK cells to activated CB-NK cells. The expression of 148 genes was significantly up-regulated at least 1 .5 fold (p < 0.05), including IFN-γ and TNF-β, and 230 were significantly down-regulated at least 1 .5 fold (p < 0.05), including CXCR3 and CXCR4, in activated CB-NK cells compared to resting CB-NK cells (Figure 10A). Notably, we confirmed that following stimulation with IL-15 activated CB-NK cells secrete significantly more IFN-γ and TNF-β than resting CB=NK cells (Figure 10B).

Discussion

Here we show that CB-NK cells impact on CBSC function by modifying their homing receptor repertoire, improving their migration and clonogenic capacities in vitro, and by enhancing engraftment in vivo. We propose a model whereby events between co-transplanted cells influence the biological efficiency and engraftment of CBSC in the BM of humanized mice. However, we also show the considerable variability that can be observed between CB units and thus the relevance of the quality of the CB units used for CBT.

We explored the effect of accessory cells on CBSC engraftment in vivo using NSG mice. Notably, our results show that the addition of CD8+ T cells, but not CD4+ T cells, increased the migration capacity of CBSC in vitro and in vivo. Higher levels of engraftment were observed in NSG mice transplanted with CBSC and resting CB-NK cells or activated CB-NK cells compared to CBSC alone. Variable levels of engraftment were observed between CB units; however, higher levels of engraftment were consistently observed in mice that received CBSC and resting CB-NK or activated CB-NK cells. The stimulus used to activate NK cells, IL-15, was selected based on the knowledge that this protein is produced in the BM and on current usage of IL-15 in clinical trials (http://clinicaltrials.gov). Nevertheless, the level of NK cell activation with IL-15 is variable and further research on the activation status of CB-NK cells, notably prior IL-15 stimulation, is needed in order to better understand whether the extent of activation could potentially explain the variability observed in the levels of engraftment in vivo. It is possible that a longer incubation time with IL-15 could lead to a more consistent activation of CB-NK cells. Although we observed variability amongst CB units in the results obtained in vitro, greater variability was observed for the in vivo experiments. The variability obtained in all in vivo experiments could potentially be greater than the variability between CB units in a clinical setting, as the CB units used in this work were research grade, thus with a lower quality standard when compared to clinical grade CB units. It will be important in the future to confirm that clinical grade CB units represent a more consistent quality of product.

The importance of the chemokine receptor CXCR4 in HSC homing to the BM is well documented. Recent reports correlate CBSC migration in vitro with engraftment in humans, providing further support for the model that HSC migration is a critical step in the successful establishment of hematopoiesis. We showed that the expression of homing receptors by CBSC was modified in the presence of activated CB-NK but not resting CB-NK cells. Of particular interest was the higher expression of CXCR4 observed by CBSC in the presence of activated CB-NK cells. Moreover, the migration capacity of CBSC assessed by in vitro transwell migration assay was also improved in the presence of activated CB-NK cells. For these assays we used a standard SDF-1 a concentration and a suboptimal concentration to assess whether activated CB-NK cells had a synergic effect on CBSC migration. A higher migration index was observed when using CBSC co-cultured with activated CB-NK cells. It is possible that the positive effect observed on CBSC migration in the presence of activated CB-NK cells is secondary to the higher levels of CXCR4 expression.

A higher number of CFU was observed for CBSC in the presence of activated CB-NK cells from either short-term or long-term cultures suggesting that activated CB-NK cells not only modify CBSC trafficking capacity but also their clonogenicity. Notably, for all the in vitro assay data presented here a 4 h co-culture between CBSC and CB-NK cells was used, however 24 h co- cultures were also performed yielding the same results. We recognize that the CBSC:NK cells ratio (1 :5) used for all in vitro experiments may or may not reflect the physiologic context. However, we chose this ratio to ensure that it would not overwhelm the in vitro models used. We attempted to assess whether the effects of activated CB-NK cells on CBSC was mediated via soluble factors or was cell contact dependent by performing CFU assays using transwells, but we were unable to pursue this approach as the transwell plates that had to be used to address this question impacted negatively on the interaction of NK cells with CBSC. However, the assays performed using blocking antibodies against beta-7 integrin, CD1 1 a, CD49d and CD49e suggest that cell-cell interaction between CBSC and NK cells via these molecules is required for the latter to have an effect on the clonogenic capacity of CBSC.

Finally, we showed by gene expression profiling analysis and ELISA that CBSC secreted high levels of CXCL9 after co-culture with activated CB-NK cells. Moreover, the microarray analysis also revealed that CBSC expressed higher messenger levels of genes that are regulated by IFN-γ and that activated CB-NK cells secreted significantly more IFN-γ and TNF-β than resting CB-NK cells. It could be that the CB-NK cells secrete IFN-γ, which in turn induced CXCL9 expression by CBSC, acting on their clonogenicity as we showed that recombinant CXCL9 increased CBSC clonogenicity. The effects of CXCL9 on CBSC phenotype and functions need further investigation as little is known of the impact of this chemokine on HSC.

The data presented here demonstrate that CB-NK cells can impact on the engraftment, homing and clonogenicity of CBSC. We therefore propose that CB-NK cells are a facilitator of CBSC engraftment in vivo. In conclusion, these results give the basis for a novel approach in the area of CBT whereby NK cell therapy or the manipulation of the composition of the cellular content of a CB unit by using cytokines such as IL-15, could be used to improve CBSC engraftment and CBT outcomes.

Claims

1 . - A method of obtaining a stem cell, wherein said stem is characterized by not being obtained by the destruction of a human embryo, with at least enhanced engraftment capacity in comparison to a control stem cell not culture in contact with a resting or activated NK cell, comprising co-culturing said stem cell in contact with at least a resting or activated NK cell.

2. - The method according to claim 1 , wherein the NK cell is an activated NK cell and wherein the resulting stem cell has an enhanced engraftment, clonogenic and migratory capacity in comparison to a control stem cell not culture in contact with said NK cell.

3.- The method according to claim 2, wherein the NK cell has been activated with IL-15.

4. - The method according to claim 3, wherein the NK cell has been activated with IL-15 for at least 2 hours.

5. - The method according to any one of claims 2-3, wherein the stem cell is co-cultured with the activated CB-NK cell for at least 4 hours.

6.- The method according to claim 1 , wherein the stem cell is co-cultured with the NK cell in the presence of IL-15.

7. - The method according to claim 6, wherein the stem cell is co-cultured with the NK cell in presence of IL-15 for at least 2 hours.

8. - The method according to any one of claims 1-7, wherein the stem cell is selected from the list consisting of mesenchymal stem cells, hematopoietic stem cells, non human embryonic stem cells, induced pluripotent stem cells, adult stem cells, mesothelial cells, hematopoietic stem cells, umbilical cord blood stem cells, or any combinations thereof.

9. - The method according to claim 8, wherein the stem cell is an umbilical cord blood stem cell (CB NK).

10.- A stem cell or stem cell population obtainable by the method according to any one of claims 1 -9.

1 1 . - A stem cell population comprising at least 80% of the stem cells as defined in claim 10.

12. - A composition comprising: a) a cell co-culture comprising a stem cell, a NK cell and IL-15, or b) a stem cell or stem cell population as defined in any of claims 10 or 1 1 . or c) a cell co-culture comprising a stem cell and an activated NK cell with IL-15.

13. - The composition according to claim 12, wherein the NK cell of step a) or c) is a cord blood natural killer cell.

14. - The composition according to any one of claims 12 or 13, wherein said composition is a pharmaceutical composition.

15.- The composition according to claim 14 further comprising an acceptable pharmaceutical carrier.

16. - A kit of parts comprising: a) at least two compositions, tubes or recipients wherein each comprises separately or in combination stem cells, CB-NK cells and IL-15, or b) at least two compositions, tubes or recipients wherein each comprises separately or in combination a stem cell and an IL-15 activated CB-NK cell.

17. - A stem cell or stem cell population according to any one of claims 10-1 1 or a composition according to any one of claims 12 to 15, or a kit of parts according to claim 16, for use in therapy.

18.- A stem cell or stem cell population according to any one of claims 10-1 1 or a composition according to any one of claims 12 to 15, or a kit of parts according to claim 16 for use in partially or completely increase, restore or replace the functional activity of a diseased or damaged tissue or organ.

19. - A stem cell or stem cell population according to any one of claims 10-1 1 or a composition according to any one of claims 12 to 15, or a kit of parts according to claim 16 for use in the treatment, amelioration or prevention of a bone marrow disease or disorder.

20. The stem cell, the stem cell population, the composition, or the kit of parts according to claim 19, wherein the bone marrow disease or disorder is selected from the list consisting of: a leukemia, a lymphoma, a myeloproliferative disease, a myelodysplasia syndrome and a plasma cell disorder, or combinations thereof

21 . - The stem cell, the stem cell population, the composition, or the kit of parts according to claim 19, wherein the bone marrow disease is a plasma cell neoplasm selected from the group consisting of multiple myeloma, plasmacytoma, macroglobulinemia, monoclonal gammopathy of undetermined significance (MGUS), or combinations thereof.

22.- The stem cell, the stem cell population, the composition, or the kit of parts according to claim 19, wherein the bone marrow disease or disorder is a hemoglobinopathy selected from the group consisting of sickle-cell disease and beta- thalassemia.

23. - A stem cell or stem cell population according to any one of claims 10-1 1 or a composition according to any one of claims 12 to 15, or a kit of parts according to claim 16, for use in the treatment, amelioration or prevention of ischemia or peripheral artery disease.

24. - The stem cell, the stem cell population, the composition, or the kit of parts according to claim 23, wherein the ischemia or peripheral artery disease is selected from the list consisting on: cerebrovascular ischemia, renal ischemia, pulmonary ischemia, limb ischemia, ischemic cardiomyopathy and myocardial ischemia or combinations thereof.

25. - The stem cell, the stem cell population, the composition, or the kit of parts according to any one of claims 23 or 24, wherein the ischemia or peripheral artery disease is the critical limb ischemia.

26. - A stem cell or stem cell population according to any one of claims 10-1 1 or a composition according to any one of claims 12 to 15, or a kit of parts according to claim 16, for use in hematopoeitic transplantation.

27. - A stem cell or stem cell population according to any one of claims 10-1 1 or a composition according to any one of claims 12 to 15, or a kit of parts according to claim 16, for use in the treatment, amelioration or prevention of stroke.

28. - A stem cell or stem cell population according to any one of claims 10-1 1 or a composition according to any one of claims 12 to 15, or a kit of parts according to claim 16, for use in the treatment, amelioration or prevention of erectile dysfunction.

29.- A stem cell or stem cell population according to any one of claims 10-1 1 or a composition according to any one of claims 12 to 15, or a kit of parts according to claim 16, for use in the treatment, amelioration or prevention of wound healing.

30.- A stem cell or stem cell population according to any one of claims 10-1 1 or a composition according to any one of claims 12 to 15, or a kit of parts according to claim 16, for use in the treatment, amelioration or prevention of diabetic neuropathy.