Method for grading then pooling mesenchymal stromal cells

Abstract

The invention pertains to methods for preparing pools of mesenchymal stromal cells for treatment of immune or inflammatory diseases, by screening cells from individual donors and including cells with high immunomodulatory properties in the pools.

Description

METHOD FOR GRADING THEN POOLING MESENCHYMAL STROMAL CELLSThe invention pertains to methods for preparing Mesenchymal stromal cells (MSCs) for treatment of immune or inflammatory diseases.Mesenchymal stromal cells (MSCs) have been extensively studied for the development of advanced therapy medicinal products (ATMPs), in particular cell and tissue engineered therapies. However, to date, only a few MSC-based cellular therapies have been approved, highlighting the complexity to transfer MSC-based products from the bench to the market. The main challenge is the high heterogeneity of MSC biological properties, which makes it difficult to standardize production processes and predict the clinical response. This variability can be related to culture conditions, be tissue-specific or donor-dependent (Frangois et al., Mol Then 2012 Jan;20(1): 187-95.). It is known that umbilical cord-derived MSCs (UC-MSCs) display higher immunoregulatory properties than MSCs derived from adult tissues (bone marrow, adipose tissue), and therefore are favored as more interesting for the treatment of immune and / or inflammatory diseases (Mebarki et al. Stem Cell Res Ther. 2021 Feb 26; 12(1): 152). However, the donor-to-donor variability of UC-MSCs is more important compared to adult tissues derived MSCs (Wang Z, Chai C, Wang R, Feng Y, Huang L, Zhang Y, et al. Single-cell transcriptome atlas of human mesenchymal stem cells exploring cellular heterogeneity. Clin Transl Med. 2021 Dec 29;11 (12):e650).Previous study performed on UC-MSCs isolated from 12 donors emphasized the variable yield of cell isolation and of their proliferative capacities (Mebarki et al., Stem Cell Res Ther. 2021 Nov 13; 12(1 ):571 ). The lack of predictive biomarkers to identify optimal donors makes it difficult to predict the therapeutic effect. Currently, the majority of products in development use MSCs isolated from a single donor, with the risk of an insufficient clinical response if used cells have poor biological properties. Several strategies have been tested to enhance MSC properties and reduce the donor-dependent heterogeneity, including hypoxic or cytokines preconditioning to normalize the immunomodulatory functions of UC-MSCs. However, transcriptomic analysis of MSCs revealed the persistence of variations in biological properties after cytokines treatment and was associated with a decrease of cell cycle division. Hypoxic conditions allowed to preserve proliferative capacitiesof umbilical cord blood-derived MSCs without standardizing their functions between donors, underlying the heterogeneity of MSCs response to hypoxia.More recently, pooling MSCs isolated from multiple donors has emerged as a new strategy to reduce the donor-dependent heterogeneity. Few protocols have been tested in clinic, including graft-versus-host disease and critical limb ischemia, using mostly bone marrow-derived MSCs. They suggested the clinical benefit of pooled MSCs from multiple donors compared with MSCs isolated from a single donor. Conversely, Hejretova et al. (Cell Tissue Bank. 2020 Mar;21 (1):119-129) showed similar immunomodulatory properties between single donors and pooled MSCs. These differences may be explained by the strategy of current protocols, which aim to pool MSCs from randomly donors. A prior selection of donors, could improve biological properties of pooled MSCs. For example, UC-MSCs pooled based on the gender of born donors, has showed differences in proliferation and cytokines expression (Kannan et al, Stem Cell Rev and Rep. 2022; 18(5): 1851-64.US2022323504A1 describes a method in which mesenchymal stem cells (MSCs) from multiple donors are pooled and the pools are used to treat inflammatory and autoimmune diseases and central nervous system disorders. A selection algorithm is applied before pooling the cells, based on a series of at least three functional tests evaluating immunomodulatory criteria. Each MSC population is scored based on the results obtained, allowing only the best-performing cells to be selected. Cells that do not meet the criteria defined by the algorithm are rejected (as implied in

[0020] ) and excluded from the pooling process. This differs from the current method, which describes the use of a single test and the possible inclusion of cells classified as being the least effective.US2023302056A1 describes a method for selecting and pooling allogeneic mesenchymal stem cells (MSCs) from at least three donors to treat and prevent COVID-19 infection and its symptoms. The teachings of this document is similar to that of US2022323504A1 .These two documents thus describe the creation of pools, but after screening using three tests and rejecting the least interesting cells.Mebarki et al (Stem Cell Research & Therapy, vol. 12, no. 1 , 2021) describe the development of an advanced therapy medicinal product (ATMP) based on umbilical cord-derived mesenchymal stromal cells (UC-MSCs) for the treatment of immune and inflammatory diseases. This document mentions that there is significantvariability between donors, which complicates product standardization, and proposes comparing cells from a single donor with pooled cells to reduce this heterogeneity (last sentence).Mebarki et al (Stem Cell Res Ther 12, 152 (2021)) is a review of the potential of umbilical cord-derived mesenchymal stem / stromal cells (UC-MSCs), particularly for the treatment of immune and inflammatory diseases. This document mentions clinical trials that have used such cells and reiterates that standardization processes should be implemented.Dominici et al (Cytotherapy. 2006 ;8(4):315-7) propose minimum criteria for defining multipotent mesenchymal stromal cells (MSCs) and establishes three essential criteria: adherence to plastic, specific expression of surface markers (CD105, CD73, CD90 in the absence of CD45, CD34, CD14 / CD11 b, CD79a / CD19, H LA-DR) and differentiation capacity into osteoblasts, adipocytes, and chondroblasts. The objective is to standardize the characterization of MSCs to improve the comparability of studies and promote advances in cell therapy.US2024182966A1 describes a method for evaluating the immunomodulatory and angiogenic capacity of mesenchymal stromal cells (MSCs) using measurable criteria such as the expression of certain genes, the secretion of certain proteins, and cell morphology. The objective is to optimize the selection and culture of MSCs to improve their clinical efficacy by reducing variability related to culture conditions and donor characteristics for the development of treatments for inflammatory and degenerative diseases.Amati et al (Stem Cell Res Ther. 2017 Jan 24;8(1 ): 14) explore the potential of mesenchymal stromal cells (MSCs) derived from umbilical cord blood for clinical use by evaluating their proliferation capacity, genetic stability, and immunomodulatory properties. The results show variability in immunosuppressive efficacy, highlighting the need for rigorous testing before any therapeutic use.The present invention provides a method for grading the immunomodulatory property of MSCs, which makes it possible to select the donors whose cells are to be included in pools. The inventors indeed assessed human UC-MSCs derived from 10 donors and were able to confirm variations in their biological properties. Based on the immunoregulatory functions of UC-MSCs in vitro, the inventors propose to grade or rank the profiles of donors as “high potential” or “not high potential” to make sure that cells from a “high potential” donor can be included in a pool with cells of“not high potential” donor(s). The inventors were also able to grade the cells in three profiles (the “not high potential” include “medium potential” or “low potential” cells). Using MSCs with a high immunomodulatory potential or property makes it possible to increase the therapeutic potential (immune properties) of the pooled cells even though the pool contains cells with a lower potential.Using the cells of one “high” donor in various pools makes it possible to increase the number of batches of ATMPs, and hence to treat more patients, with a higher expectation of therapeutic success. This is of interest, as only a handful (generally about 20%) of donors present this “high” potential property with regards to the cell’s immunomodulatory property.The proposed method thus makes it possible to pool MSCs using tests that are simpler than the previous ones, but also solves a technical issue that is to increase the number of cells in a pool by using cells with lower potential. Indeed, contrary to the teachings of US2022323504A1 and US2023302056A1 , the claimed method makes it possible to retain and use the cells of poor donors, which can be retained when mixed with cells of best donors, while these documents eliminate the cells of poor donors. In particular, it is shown that the cells with “high” potential can also be mixed with only cells with “low” potential, at a 1 :2 or 1:5 ratio, and that such pooling improves the immunomodulatory properties of the lowest cells to the same level as the highest cells. The use of MSCs from one donor graded as high in different pools will offer the possibility of (i) optimizing the use of “high” potential cells, (ii) increasing the number of clinical batches, and hence (iii) treating more patients with a higher expectation of immunoregulatory effects.In 2006, the International Society for Cellular Therapy (ISCT) established the following unified and minimal criteria to define MSCs (Dominici et al,. Int Soc Cell Ther position statement Cytotherapy. 2006;8(4):315-7).Plastic-adherent cellsExpression of the surface markers CD73, CD90 and CD105, but not the hematopoietic markers CD45, CD34, CD14, CD11b, CD19, CD79a or HLA-DR- Trilineage mesenchymal differentiation capacity into osteoblasts, adipocytes and chondrocytes.It is reminded that the immunomodulatory properties of mesenchymal stromal / stem cells (MSCs) are a key aspect of their therapeutic potential. Their immunomodulatory characteristics includeImmunosuppression: They can inhibit naive and memory T-cell responses and communicate with antigen-presenting cells, thereby modulating immune responses.Regulation of Immune and Inflammatory Responses: they secrete cytokines, chemokines, signaling molecules, and growth factors, which effectively contribute to the regulation of immune and inflammatory responses.Modulation of Immune Cells: they can interact with innate and adaptive immune systems via cell-to-cell interactions and immunomodulatory / regenerative factors, affecting the phenotype and functional properties of monocytes / macrophages, dendritic cells, T cells, and B cells.In summary, in the view of their immunomodulatory properties, MSCs have a therapeutic potential for the treatment of a variety of immune-mediated diseases, including multiple sclerosis (MS), osteoarthritis, rheumatoid arthritis (RA), collagen- induced arthritis, Type 1 diabetes mellitus, systemic lupus erythematosus, inflammatory bowel diseases (including Crohn’s disease and ulcerative colitis), inflammatory airway & pulmonary diseases (such as obstructive lung diseases (COPD), asthma, idiopathic pulmonary fibrosis (IPF), acute respiratory distress syndrome (ARDS), Bronchial pulmonary dysplasia), graft versus host disease (GvHD), and allergic disorders. They can also be used in the treatment of infectious diseases (Sharma et al, World J Stem Cells. 2021 Jun 26; 13(6): 568-593). One can also cite the diseases mentioned in Hoang etal (Signal Transduct Target Ther. 2022; 7: 272), who cites cardiovascular diseases (see also Hare, J.M. et al. J. Am. Med. Assoc. 308, 2369-2379 (2012)), digestive system diseases (see also above), liver diseases (liver failure, cirrhosis, cancer, alcoholic liver disease, nonalcoholic fatty liver disease (NAFLD), and autoimmune liver disease (ALD)), and indicates that bone marrow MSCs are good candidates for brain and spinal cord injury treatment. Adipose Tissue MSCs (AT-MSCs) are good candidates for reproductive disorder treatment and skin regeneration, and UC-MSCs are good candidates for pulmonary disease and acute respiratory distress syndrome treatment.The invention thus relates to a method for evaluating and grading the immunomodulatory property of a population of human mesenchymal stromal cells (MSCs), comprising a. Revealing immunomodulatory properties of mesenchymal stromal cells cultured in a resting state and / or primed in pro-inflammatory conditions b. Obtaining a numerical value associated with the immunomodulatory property of the cells c. Grading the immunomodulatory property of the population of cells as high, medium or low, wherein a grade “high” or “medium” is assigned when the immunomodulatory property of the population of the cells is above the median, and wherein a grade “low” is assigned when the immunomodulatory property of the population of the cells is below the median, wherein the median has been obtained from a series of numerical values associated with the immunomodulatory properties of multiple populations of mesenchymal stromal cells from multiple subjects.This method thus makes it possible to select donors the cells of whom will be included in pools, and in particular to identify donors the cells of whom have a high immunomodulatory potential and ability. Such method is performed on an aliquot of MSCs isolated from a donor then cultured in vitro.Thus pools of MSCs can be prepared with prior selection of donors (and thus not randomly), in order to obtain a composition where the functions of cells whose immunomodulatory properties are low are improved. The donors whose cells have high immunomodulatory properties are qualified “high” donors.The MSCs are first cultured either in a resting state or in pro-inflammatory conditions. In particular, the cells may be cultured in the presence of pro- inflammatory cytokines. Pro-inflammatory cytokines are signaling molecules that are involved in the inflammatory response. They are produced by various cells, notably immune cells, and help to regulate the immune response, promote inflammation, and recruit immune cells to the site of infection or injury. As pro-inflammatory cytokines, one can cite Tumor Necrosis Factor-alpha (TNF-a), lnterleukin-1 (IL-1) (notably IL-1ct or IL-113), lnterleukin-6 (IL-6), lnterleukin-8 (IL-8), Interleukin-12 (IL-12), Interleukin-17 (IL-17), Interferon-gamma (IFN-y), or Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF).Culture in the presence of the pro-inflammatory cytokines “primes” the MSCs thereby initiating, enhancing or modifying their response to subsequent stimuli. In particular, such priming would make the cells able to interact with cells of the immune system. Immunomodulation ability of the cells can thus be determined. Such response can increase cytokine production (such as IL-6, IL-8, IL-10 (immunosuppressive effect), various Growth Factors, Chemokines, and Signaling Molecules (in particular VEGF that plays a crucial role in cell engraftment, neovascularization, and wound healing), Intercellular adhesion molecule-1 (ICAM- 1) and vascular cell adhesion molecule 1 (VCAM-1) or IDO (Indoleamine 2,3- dioxygenase), the presence of which being associated with the suppression of inflammatory processes).The immunomodulating property of the primed MSCs can thus be evaluated in any assay that is known in the art. One can cite:- Cytokine Production Assays such as ELISA (Enzyme-Linked Immunosorbent Assay) for measuring amount of a give cytokine, or of multiple cytokines, in Multiplex Cytokine Assays,- Flow Cytometry, to identify and quantify expression of surface markers indicative of activation, differentiation, or specific cell subsets, or of intracellular cytokine (Intracellular Cytokine Staining),- Phagocytosis and Killing Assays, to evaluate phagocytic activity, or ability to kill pathogens,- Proliferation Assays of immune or inflammatory cells, such as BrdU / EdU Incorporation Assay, using thymidine analogs, CFSE Dilution Assay, using CFSE (a fluorescent dye) and analysis by flow cytometry,- Gene Expression Analysis such as qPCR (Quantitative Polymerase Chain Reaction), RNA-Sequencing,- Functional Assays, such as Mixed Lymphocyte Reaction (MLR), where the primed or non-primed cells are co-cultured with allogeneic T-cells, and T-cell proliferation is measured, or Cytotoxicity Assays, to measure the ability of the primed cells to kill target cells.In one embodiment, the immunomodulatory property of the cells is assessed by the expression of markers, notably by ELISA, RNA-sequencing or flow cytometry. In this embodiment, it is preferred to assess expression of at least one marker selected from IDO, Intercellular Adhesion Molecule-1 (ICAM-1 / CD54), programmed death ligand 1 (PD-L1 / CD274), vascular cell adhesion molecule 1 (VCAM-1 / CD106), CD200. Assessment of production of IDO, ICAM-1 or VCAM-1 is preferred. Amount of the molecule is measured before (basal or resting state) and after (primed state) culture in presence of the pro-inflammatory cytokines.In another embodiment, the immunomodulatory property of the cells is assessed by Mixed lymphocyte reaction (MLR). MLR assay can be performed on MSCs both in a resting state (NT) or after priming for 48 h with IFNy, TN Fa and IFNy + TNFa. Briefly, peripheral blood mononuclear cells (PBMCs) pooled from 10 allogenic donors can be co-cultured with MSCs at various (0:1 (control), 1 :5, 1 :10, 1 :30, 1 :100, 1:300 and 1 :1000) MSCs:PBMC ratio with a constant amount of PBMC (3 * 105) and a decreasing amount of UC-MSCs from 6 x 104down to 3 * 102. PBMCs are then labeled with labeled antibodies against CD3, CD45 and 7-AAD and analyzed by flow cytometry. It is reminded that MSCs have immunomodulatory properties and would thus inhibit proliferation of the lymphocytes in the PBMCs (as compared to proliferation observed with the control).In particular, Mixed Lymphocyte Reaction assay is performed with MSCs in a resting state, at a 1:30 MSCs:PBMC ratio (using 3 x 1O5PBMCs).Any above method makes it possible to obtain a numerical value. When performing MLR, the numerical value may be the percentage of inhibition of lymphocyte proliferation in presence of MSCs (as compared to the proliferation in the absence of MSCs). When measuring expression of a marker, the numerical value may be the percentage of increase or decrease of expression of the markers (as compared to the expression measured in cells in the native state).In order to grade or classify the immunomodulatory properties of the cells of a given donor, the numerical value obtained is compared to a set of values that has been obtained from prior analysis of a series of MSCs from multiple donors. It is advisable to use a series of 10 or more donors, in order to obtain a diversity ofdonors, so that the used donors include donors with cells of high immunomodulatory properties.From the different values obtained from the analysis of the immunomodulatory properties of the MSCs from multiple donors, it is possible to determine a median value, and quartile (first quartile, third quartile) values. Such median and quartile values are used in the method herein disclosed.When the numerical value obtained for the tested MSCs is below the median value, a grade “low” is assigned to the cells. This indicates that the MSCs of the donor have low immunomodulatory ability. Such cells are not to be used alone, but may still be included in a pool with cells with a higher grade.When the numerical value for the tested MSCs is above the median, the cells are graded “medium” or “high”. This indicates that the cells could be used as such (alone) for treating a patient. However, it is preferred to use the cells in pools with cells of a “low” grade. This makes it possible to prepare multiple and large batches of AT Ps and increase the number of treated patients, with the pools of cells.In another embodiment, the grade “high” is assigned when the numerical value associated with the immunomodulatory property of the cells is within the last quartile (quartile with the higher values), and wherein the grade “medium” is assigned when the numerical value associated with the immunomodulatory property of the cells is within the third quartile (between the median and the third quartile) of the series of numerical values associated with the immunomodulatory properties of the multiple populations of mesenchymal stromal cells from multiple donors.This embodiment makes it possible to select the best (“high”) cells from a donor to make sure that they are included in pools of MSCs used for preparing ATMPs.In another embodiment, the grade “high” is assigned when the numerical value associated with the immunomodulatory property of the cells is above the sum of the median and the interquartile value, and wherein the grade “medium” is assigned when the immunomodulatory property of the population of the cells is above the median and below the sum of the median and the interquartile value. Using this method of grading the cells may sometime be more appropriate, providing three grades (high, medium and low) and giving more options for pooling the cells.It is understood that the purpose is to classify the cells in comparison to each other. There may be some variability in the median and interquartile values when different series of MSCs from multiple donors. The examples provide an illustrationof a value of the numbers that can be used when MLR is used as an assay fortesting the immunomodulatory properties of the MSCs.In the preferred embodiment, the mesenchymal stromal cells were isolated from human umbilical cord (umbilical cord-derived MSCs (UC-MSCs)). MSCs can be isolated from Wharton's jelly by enzymatic methods or explant methods.In another embodiment, the mesenchymal stromal cells have been obtained from bone marrow. The mononuclear fraction is obtained through bone marrow aspiration, possibly followed by density gradient centrifugation. MSCs adhere to plastic and can be further separated from other cells in the mononuclear fraction.In another embodiment, the mesenchymal stromal cells have been obtained from adipose tissue. Adipose tissue can be obtained through liposuction, and MSCs can be isolated by enzymatic methods or explant methods.Using the grading system herein disclosed makes it possible to implement a method for preparing a composition of mesenchymal stromal cells, comprising: a. Providing mesenchymal stromal cells from at least two donors b. Separately testing the mesenchymal stromal cells of a., with regards to their immunomodulatory properties, c. Selecting first mesenchymal stromal cells from a first donor, wherein the cells present high immunomodulatory properties d. Selecting second mesenchymal stromal cells from a second donor, wherein the cells present medium or low immunomodulatory properties e. Mixing the first and second selected mesenchymal stromal cells to obtain a pool of mesenchymal stromal cells.This composition (or pools) of MSCs can be designated as “heterogenous”, as it contains cells from different donors.In b), the cells of each donor are separately tested, and can be graded according to their immunomodulatory properties, with grades being high, medium and low. This makes it possible to identify and select the donor the cells of whom have high immunomodulatory properties, and use the cells of this donor together with cells of another donor (potentially two other donors) who have cells of medium or low immunomodulatory properties.MSCs from a “high” donor are mixed with MSCs from at least one “low” or “medium” donor.When cells for one high donor are mixed with cells from one “medium” donor and cells from one “low” donor, the ratio may be 1 :1 :1 (same number of cells from each donors).When cells for one high donor are mixed with cells from one “medium” donor, the ratio may be comprised between 1 :5 and 1:1, preferably 1:2 and 1 :1.When cells for one high donor are mixed with cells from one “low” donor, the ratio may be comprised between 1 :5 and 1 :1 , preferably 1 :2 and 1 :1.Aliquots or bags of MSCs from each donor are amplified then can be either:- individually frozen and pooled just before use.- individually frozen and pooled just before the step of work cell stock.- pooled and frozen just before the step of master cell stock.Master cell stocks are prepared with the cells of each donor.It is to be noted that, to date, it is generally not recommended to amplify MSCs more than a few times (about 4-5 passages) without the cells losing their properties (Mebarki et al, Stem Cell Research & Therapy, 2021). Hence, once a “high” donor has been identified, the clinical value of the cells is very high and it is preferred to advise to save them.Using the cells of the pools for treatment of a patient would make it possible to obtain adequate clinical response, as shown in the examples. Indeed, the inventors showed that the presence of MSCs with high immunomodulatory properties, in a pool with other cells that do not have the same properties, makes it possible to obtain an adequate global immunomodulatory biological effect.This would make it possible to observe a reduction in the variability of clinical responses, observed when MSCs which are derived from a single donor (consequently, each time the donor is changed, the response is different, making it difficult to anticipate), and to avoid the risk of inefficiency or low efficacy associated with the use of MSC derived from a single “low” donor or from a pool of several low and / or medium donors (the prior selection herein disclosed thus sets the method apart from prior art describing making pools, without prior donor selection).In a preferred embodiment the testing and grading of the mesenchymal stromal cells is performed by the method disclosed above.After mixing the cells of multiple donors, multiple aliquots of the cells of the pool can be prepared. The number of cells in each aliquot is such that between 105and 107cells / kg can be administered to a patient, using one aliquot or several aliquots.The pool or the aliquots can be frozen -in DMSO 5 - 10% and then stored for cryopreservation in nitrogen at a temperature below -80°C, and generally at -120°C. It is advisable to prepare various master cell stocks (master banks) from each donor. Then an aliquot of the master cell stock from high donor and another from low and / or medium donor(s) may be thawed, cultured, expanded, then pooled just before freezing and cryopreservation to prepare the work cell stock. When there is a need of use, an aliquot of the work cell stock is thawed, the cells can be used for the treatment of one or more patients.The invention thus also relates to a pool of mesenchymal stromal cells, wherein the pool contains cells from at least two donors, wherein the cells of one donor are graded as having high immunomodulatory property, and wherein the cells of the other donor(s) are graded as having medium or low immunomodulatory property.The invention also relates to the pool as disclosed as a medicament or for use thereof as a medicament. One can also cite use of the pool as disclosed for the preparation of a medicament.In particular the pool of mesenchymal stromal cells, wherein the pool contains cells from at least two donors, wherein the cells of one donor are graded as having high immunomodulatory property, and wherein the cells of the other donor(s) are graded as having medium or low immunomodulatory property, can be used for the treatment of immune or inflammatory diseases, or other diseases as indicated above.A method for treating a patient in need thereof, comprising administering cells of a pool of mesenchymal stromal cells as herein disclosed to the patient can thus be implemented. In particular, the cells are provided at a dose comprised between 105 / kg to 107 / kg to the patient, in one or more administrations.It is reminded that cells can be counted by any method known in the art: Malassez cell count, automated cell count with or without viability marker, flow cytometry with a specific marker.As indicated above, it is possible to prepare a mesenchymal stromal cells for therapeutic use, by thawing a frozen pool or aliquot herein disclosed, and culturing and / or expanding the thawed cells. The cultured or expanded cells can then be used for one or more administration to a patient. The pools can also be used after thawing, without expanding cells.FIGURESFigure 1 : Description of immunomodulatory properties of each of the 10 donors. A. Median (left graph) and individual (right graph) expression of IDO (%) at basal state (NT) and after pro-inflammatory priming by I FNy and IFNy + TNFa. UC 185 and UC 186 were not treated with IFNy + TNFa and are presented in dotted lines. B. Inhibition of T-lymphocyte proliferation (%) assessed by MLR assay at basal state (NT) (ratio UC-MSCs:PBMC 1 :30). n=1 / donor.Figure 2: Constitution of UC-MSCs pools with prior selection of donors based on their immunoregulatory functions. A. The process of constitution of UC-MSCs pools. Comparison of time doubling (TD) between B. the theoretical mean of individual donors and the pool.Figure 3: Expression of adhesion surface markers in UC-MSCs isolated from individual donors versus pool. Expression (%) of A. CD44, B. ALCAM / CD166, C. CD146, D. ICAM-1 / CD54, E. CD200, F. VCAM-1 / CD106 and G. PDL-1 at basal state (non-treated) and after pro-inflammatory treatment with IFNy and IFNy+TNFa.Figure 4: Expression of markers of immunogenicity in UC-MSCs isolated from individual donors versus pool. Expression (%) of A. HLA-DR (individual donors versus pool), B. HLA-DR (theoretical mean of individual donors versus pool), C. CD86, D. CD40 at basal state (non-treated) and after pro-inflammatory treatment with IFNy and IFNy+TNFa.Figure 5: Expression of IDO and IFNy-Receptor in UC-MSCs isolated from individual donors versus pool. Expression (%) of A. IDO (individual donors versus pool) B. IFNy-Receptor and C. IDO (theoretical mean of individual donors versus pool) at basal state or under pro-inflammatory conditions (treatment with IFNy or IFNy+TNFa).Figure 6: suppression of T-cells expansion in vitro with a ratio -MSCs:PBMC.A. untreated MSCs with a decreased UC-MSCs: PBMC ratio. B. MSCs at basal state or under pro-inflammatory conditions with a UC-MSCs:PBMC =1:10 ratio. C. theoretical average of the three donors versus pool. D-l. Further comparison of thetheoretical mean of individual donors versus the pool. (n=5 independent experiences per donor, pool, and per condition).Expression of IDO in UC-MSCs isolated from high and low donors versus pool of 2 or 3 donors. Expression (%) of A. IDO of cells from individual donors and 2 donors (2D) pools at different ratio B. IDO of cells from 2D and 3D pools. C-E. Comparison of T-cell suppression between individual high and low donors versus the pool 2D with a decreased UC-MSCs: PBMC ratio of 1 / 10; 1 / 30; 1 / 100; 1 / 300. F-H. Comparison of the theoretical mean of individual donors versus the pool 2D. I-K. Comparison of T-cells suppression between pools 2D and 3D. (n=5 independent experiences per donor and per condition and n=3 independent experiences for pool 2D).EXAMPLESExample 1. Material and methods1. UC-MSCs isolation and expansionUCs were collected from 10 healthy donors, according to the French regulation. UCs collection, UC-MSCs isolation and expansion were performed as previously described (Mebarki et al, Stem Cell Res Ther. 2021 Nov 13; 12(1):571).2. Preparation of UC-MSCs poolCells isolated from single donors were thawed between passages 2 and 4 maximum, washed, enumerated then seeded at a density of 1000 cells / cm2for each donor, in 75cm2culture flasks using a GMP-compliant complete culture medium composed of NutriStem® MSC XF Basal Medium (Biological Industries, Ref 05-200- 1A) + Nutristem® MSC XF Supplement Mix (Biological Industries, Ref 05-200-1 U) + 5% irradiated platelet lysate (PL) MultiPL100’i (Macopharma, Ref BC0190032) + sodium Heparin 2 lU / mL (Panpharma, Ref 5520508). UC-MSCs were harvested using a recombinant trypsin when they reached 80% of confluence. Harvested cells from each single donor were pooled in pools of 2 (2D) or 3 donors (3D) then used for adequate analysis. UC-MSCs morphology was measured using the Incucyte® S3 and analyzed on the Incucyte® S3 Live — Cell Analysis system (Sartorius). The morphology was assessed in single donors and in the pool, in the basal state.3. Cell proliferation assessmentUC-MSCs from individual donors and from pools were enumerated after each passage using a manual Malassez counting Chamber. Cells viability was assessed using an automatic cell counter EVE Plus 1 (Ozyme). Time Doubling (TD) andPopulation Doubling (PD) were determined after each passage according to the formulae (T x log(2)) / (log Y - log X) and log(Y / X) / log(2) respectively (X: number of cells originally seeded, Y: final number of cells, T: duration of culture in hours).4. Priming with pro-inflammatory cytokinesIn order to assess the immunomodulatory properties of UC-MSCs, an inflammatory environment was created in vitro to mimic that seen in patients. UC- MSCs from single donors and pools were treated with the pro-inflammatory cytokines IFNy 10 ng / mL and IFNy+TNFa 10+10 ng / mL, for 48h. A non-treated (NT) condition was used to assess UC-MSCs in the basal state. After conditioning with cytokines, cells were harvested using a recombinant trypsin EDTA solution (Life Technologies, Ref 12563011), washed and suspended in the appropriate medium depending on the analysis.5. UC-MSCs phenotypeUC-MSCs phenotype was assessed for each single donor then for the pool, at basal state (NT) and after pro-inflammatory priming, as previously described (Mebarki et al., Stem Cell Res Then 2021 Nov 13; 12(1):571). Briefly, cells were suspended in 100 pL PBS / albumin 1 % and stained concomitantly with several antibodies divided in two panels, for 15 min at 4°C, protected from the light. Cells were washed in 1 ml PBS / Albumin 1%, centrifuged at 1500 RPM for 5min and suspended in 300 pL PBS / albumin 1%. Negative controls were non-stained cells or Fluorescence Minus One for CD31, CD14 and CD45 antibodies. The acquisitions were performed with Attune NxT™ Thermofisher® Flow Cytometer and analyzes were performed using Attune NxT software.6. Expression of activation markersThe expression of activation markers was evaluated on UC-MSCs of single donors then on pooled cells, in the basal state and after priming. The expression of Indoleamine 2,3-dioxygenase (IDO), Inter Cellular Adhesion Molecule-1 (ICAM- 1 / CD54), Programmed Death-Ligand 1 (PD-L1 / CD274), Vascular Cell Adhesion Molecule 1 (VCAM-1 / CD106), CD200, INFy-Receptor (INFy-R / CD119) and TNFa- Receptor II (TNF-RII / CD120b) were assessed according to the protocol previously described (Mebarki et al., Stem Cell Res Then 2021 Nov 13; 12(1 ):571 ). Acquisitions were performed using Attune NxT™ Thermofisher® Flow Cytometer and analyses using Attune NxT™ software.7. Mixed Lymphocyte Reaction (MLR)MLR potency assay was performed according to Nicotra et al. Stem Cell Res Ther. 2020 Oct 1 ;11(1):426. MLR assay was performed on UC-MSCs both in a resting state (basal state) (NT) and after pro-inflammatory conditioning for 48 hours. Peripheral blood mononuclear cells (PBMC) pooled from 10 healthy donors were cocultured with UC-MSCs from each donor or with UC-MSCs pooled from 3 donors. The ratio of UC-MSCs: PBMC 0:1 (control), 1 :10, 1:30, 1 :100, 1 :300 and 1 :1000 were used.8. Statistical analysisStatistical analyses were performed using GraphPad PRISM® 10.1.2 software with appropriate tests, notably after assessment for non-normal distribution. Descriptive data are expressed as median [min - max] and all other values are expressed as mean ± standard deviation. A minimum of 95% confidence interval was established for significance. A p-value < 0.05 was considered statistically significant and are indicated on the figures as *: < 0.05, **: < 0.01, ***: < 0.001, ****: < 0.0001 . Kruskal-Wallis test was used to compare 10 donors and Tukey’s test was used for multiple comparison. Alternatively, Dunn’s multiple comparison test was used to compare 10 donors, the Spearman test was used for correlation analysis, the Mann-Whitney test was used for comparison between two groups and the Tukey’s test was used for multiple comparison of more than two groupsExample 2. Results1. Characteristics of collected UCsUCs were collected from 10 healthy mothers, aged from 27 to 37 years (Table 1). The median of delivery was 40.1 W[39 - 41 W], the delivery was vaginal for 9 / 10 and caesarian for 1 / 10, the sex ratio of newborns was 4 females and 6 males (F / M = 0.67), with a median weight of 3480 g [2910 - 4200 g], UCs median weight was 22.0 g [15 - 43 g]; nine of them were manipulated within 1 day and one within 3 days.Isolated cells presented morphological and phenotypic properties of UC-MSCs as previously described (data not shown) (Mebarki et al., Stem Cell Res Ther. 2021 Nov 13; 12(1 ):571 ). The total number of isolated cells at passage 0 (P0) was not correlated with the weight of the UCs. The number of isolated UC-MSCs per gram of UC was not different depending on the gender of the babies, or on the delivery method.Table 1. Characteristics of collected UCs (n = 10 donors). UC 5A: UC used in the clinical trial NCT04333368. NA: not available, g: grams, d: days, w: weeks, y: years. 2. UC-MSCs immunomodulatory functions are donor-dependentImmunomodulatory properties of UC-MSCs derived from each individual donor were analyzed. In order to limit the use of the master and work cell stocks (MCS andWCS), descriptive analysis was performed on 10 donors with n=1 independent experience per donor, . First, the expression of IDO, a key protein involved in MSC immunomodulatory function, was assessed, at basal state (non-treated, NT) and in pro-inflammatory conditions (Figure 1A). IDO was lowly expressed in non-treated UC-MSCs (0.92% [0.04 - 18.68]) and was induced after priming with IFNy (69.28% [1.28 - 87.38], p=0.0022) and IFNy+TNFa (67.22% [2.01 - 92.48], p=0.0017). Even after pro-inflammatory priming, an important heterogeneity was observed between donors, with IDO expression > 60% for seven donors and < 30% for three donors, including one donor with an IDO expression < 2%.To confirm this variability and determine whether all collected UCs are useful in clinic and identify the best ones, a MLR potency assay previously validated as a GMP- compliant Quality Control for UC-MSCs-based ATMPs was performed (Nicotra et al., Stem Cell Res Ther. 2020 Oct 1 ;11 (1):426). To mimic and reproduce the pharmaceutical manufacturing and clinical qualification of an AT P, MLR assays were performed on UC-MSCs at basal state (non-treated condition). The median inhibition of T cells proliferation with ratio 1 :30 was 53.1 % [35.5 - 96.1 %] and was used as a specification to validate the conformity of MLR assay (Figure 1B). Based on this parameter, 5 UCs were compliant (> 53.1%) including UC185 (96.1 %), UC180 (94.7%), UC190 (76.7%), UC191 (59.5%) and UC189 (55.0%) and 5 were out-of-specifications (OOS) (< 53.1% corresponding to the specification) including UC186 (51.1%), UC5A (49.3%), UC184 (42.1%), UC179 (36.5%) and UC187 (35.5%). Then, the interquartile range was used to distinguish 2 profiles within the compliant products: UC185 and UC180 displayed a high capacity (> 81.2%) to inhibit T cell proliferation whereas UC190, UC191 and UC189 presented a medium inhibitory function. All OOS products were classified as UCs with low immunomodulatory potential. Of note, neither the IDO expression nor the suppression of T-cell proliferation were associated with mother’s age, delivery term, baby’s gender and weight, or UC weightBased on these data, the MLR functional assay was used to distinguish 3 profiles of UC-MSCs immunosuppressive functions and potential: high, medium and low.3. Constitution of UC-MSCs poolTo better assess the donor-dependent variability of UC-MSCs biological properties, analysis on UC-MSCs isolated from UCs of each donor profile wasperformed (minimum n=3 / donor): high (UC180), medium (UC190) and low (UC184) that we named UC-MSCshigh, UC-MSCsmediumand UC-MSCsl0Wrespectively. Then, properties of UC-MSCs from individual donors were compared to a pool of UC-MSCs from these 3 donors (UC-MSCsp°o1). UC-MSCsp°o1consisted of UC-MSCshigh, UC- MSCsmediumand UC-MSCslowat ratio 1 :1 :1 (Figure 2A).No change in cell morphology was observed in the pool. UC-MSCs both from individual donors of each profile (UC-MSCshigh, UC-MSCsmediumand UC-MSCsl0W) and from the pool expressed mesenchymal surface markers (CD90, CD73, CD105, CD29) and did not express hematological neither endothelial markers (CD45, CD14, CD31) before and after the pro-inflammatory treatment in the basal state (not shown).As the manufacturing of cell based-therapies is time and cost consuming, MSCs proliferative potential in vitro is a critical parameter for donor selection, the theoretical mean doubling time (TD or DT) of UC-MSCs from each donor was similar to the TD of pooled UC-MSCs in non-treated and treated conditions (Figure 2B).The doubling time (DT) (h) was assessed between passages 3 and 4. In the basal state, the DT of UC-MSCsl0W(42.5±1 1.8h) was slower than of other donors (UC-MSCshigh: 31.5±15.5h, p>0.05; UC-MSCsmedium: 25.2±3.6h, p=0.0126) as well as UC-MSCsp°01(27.8±6.6h, p=0.0362) (Figure 2B). As the production process may include MSCs activation with cytokines, we also evaluated their expansion after stimulation with IFNy and IFNy+TNFa. After priming with IFNy, the DT of UC- MSCs10™ (42.8±17.1 h) was still significantly slower than of UC-MSCsmedium(26.1±3.2h, p=0.0165) and UC-MSCsp001(27.2±6.2h, p=0.0234). Finally, treatment with IFNy+TNFa showed lower proliferation of UC-MSCslow(49.6±18.7h) compared with UC-MSCshigh(32.3±9.7h, p=0.0163), UC-MSCsmedium(30.4±4.6h, p=0.0043) and UC-MSCsp°o1(33.0±11 ,3h, p=0.0139) (Figure 2B). As indicated, the theoretical mean of the DT of individual donors was similar to the DT of pooled UC-MSCs in nontreated and treated conditions. Taken together, these results showed that pooling UC-MSCs reduces the donor-dependent heterogeneity of the proliferation capacities.4. Pooling UC-MSCs allows to standardize and homogenize the expression of adhesion surface markersDespite the growing interest of MSCs paracrine properties and the use of their extracellular vesicles, cell-to-cell interaction is still a key parameter to guarantee afull efficacy of MSCs immunosuppressive functions (21,22). Thus, the expression of several markers described to be involved in MSCs cell-to-cell direct mechanisms was assessed. At basal state (non-treated) UC-MSCs from each donor and from the pool expressed several adhesion molecules including CD44, activated-leukocyte cell adhesion Molecule (ALCAM / CD166), the intercellular adhesion molecule-1 (ICAM- 1 / CD54), melanoma cell adhesion molecule (MCAM / CD146) and CD200.The expression of CD44 was similar between individual donors and compared with pooled UC-MSCs, in non-treated condition as well as after pro-inflammatory treatment (Figure 3A). ALCAM / CD166 showed donor-dependent heterogeneity in non-treated UC-MSCs with increased expression in UC-MSCsmedium(83.1 ±18.6%) compared to UC-MSCshigh(32.1 ±30.8%, p=0.0253) and UC-MSCslow(43.9±8.5%, p>005) (Figure 3B). Pooling the cells resulted in an increased expression of ALCAM / CD166 (81.1±7.1%) that became similar with UC-MSCsmediumand higher than UC-MSCshi0h(p=0.0186) and UC-MSCsl0W(p>0.05). In pro-inflammatory conditions, the expression of ALCAM / CD166 was still donor-dependent and was increased in pooled UC-MSCs (Figure 3B). Similar results were observed with MCAM / CD146, with increased expression after pooling UC-MSCs in the basal state as well as in pro-inflammatory conditions (Figure 3C). The expression of ICAM- 1 / CD54 in the basal state was higher in UC-MSCshigh(85.1±13.5%) than in UC- MSCsmedium(28.4±17.5%, p=0.0004) and UC-MSCsl0W(59.3±44.7%, p>0.05). Pooling the cells did not increase the lowest expression of ICAM-1 / CD54, whereas pro-inflammatory treatment increased the expression to > 95% in individual donors and the pool (Figure 3D). The expression of CD200 was also higher in UC-MSChighin the basal state and remained similar after pro-inflammatory treatment. Pooling UC-MSCs increased CD200 expression comparing to UC-MSCsmediumand UC- MSCs10” but was not significant (Figure 3E).The vascular cell adhesion molecule-1 (VCAM-1 / CD106) and programmed cell death protein 1 (PD-L1) were not expressed in the basal state but were stimulated after pro-inflammatory treatment (Figure 2F-G). After treatment with IFNy+TNFa, VCAM-1 / CD106 was highly expressed in UC-MSCsmediumwhereas PD-L1 increased in UC-MSCshighand UC-MSCsl0W. Pooling the UC-MSCs (UC-MSCsp°o1) resulted in an increase in the lowest expression of VCAM-1 / CD106 and PD-L1 but was still not significant compared to each individual donor.Taken together, these results highlight the existence of an important donor-to- donor variability of the expression of adhesion surface markers, which tends to be standardized by pooling UC-MSCs.5. The low immunogenicity of UC-MSCs is still not modified in the pooled productNeither the UC-MSCs of individual donors nor of the pool expressed H LA-DR in the basal state. After I FNy priming, H LA-DR expression was strongly increased in UC-MSCshighcompared with UC-MSCsmediumand UC-MSCslow(26.3±11.1% vs 2.4±1.7% vs 1 9±1.6%, p<0.0001) (Figure 4A). Similar results were observed with IFNy+TNFa (13.9±5.0% vs 0.8±0.7% vs 1.0±0.9%, p<0.01) (Figure 4A). Interestingly, after I FNy conditioning H LA-DR expression was higher in UC-MSCsp°o1than in UC-MSCsmediumand UC-MSCsl0W(12.4±4.1% vs 2.4±1.7% vs 1.9±1.6%, p<0.05), but was still lower than in UC-MSCshigh(12.4±4.1 % vs 26.3±11.1%, p<0.01) (Figure 4A). The expression of HLA-DR by UC-MSCsp001was similar with the theoretical mean of individual donors, after treatment with I FNy (12.4±4.1 vs 10.18±13.3%) and IFNy+TNFa (6.0±5.1% vs 5.2±7.0%) (Figure 4B). In parallel, the expression of CD40 and CD86 co-stimulatory molecules was also heterogeneous between donors but presented similar profiles between UC-MSCsp001and UC- MSCshigh(Figure 4C). These results demonstrate that allogeneic UC-MSCs pooled from multiple donors do not induce a cumulative immunogenic effect, which is encouraging for the clinical use of pooled UC-MSC-based ATMPs as an allogeneic product.6. IFNy-stimulated and pooled UC-MSCs enhance the lowest expression of IDOIDO was not expressed in the basal state but was enhanced in pro- inflammatory conditions, with donor-dependent variability (Figure 5A). After treatment with I FNy and IFNy+TNFa, IDO expression was significantly higher in UC- MSCs^ than in UC-MSCsmediumand UC-MSCsl0W(IFNy: 75.1±12.1 % vs 18.5±13.4% vs 10.1±8.7%; p=0.0003 and p<0.0001 respectively; IFNy+TNFa: 83.5±9.0% vs 39.8±26.4% vs 13.0±9.7%; p=0.0048 and p<0.0001 respectively; Figure 5A). IDO expression was significantly higher in UC-MSCspool compared with UC-MSCsl0W(IFNy: 46.4±23.0% vs 10.1±8.7%, p=0.0217; IFNy+TNFa: 63.8±24.6% vs 13.0±9.7; p=0.001 ; Figure 5A). Interestingly, these differences were observed despite the uniform expression of IFNy-Receptor (IFNy-R, CD119) for each donor, as well as for pooled UC-MSCs (Figure 5B). Moreover, the IDO expression by UC-MSCsp°o1wassimilar to the theoretical mean of I DO expression by the three donors, after treatment with IFNy (46.4±23.0 vs 34.6±32.3%) and IFNy+TNFa (63.8±24.6% vs 45.4±34.2%) suggesting that pooling UC-MSCs results in average IDO expression thus, reducing the inter-donor variability (Figure 5C).7. Pooling UC-MSCs increases their potential to inhibit T cell proliferationIn previous work, it was demonstrated through a MLR potency assay (Nicotra et al., Stem Cell Res Ther. 2020 Oct 1 ; 11(1):426) the capacity of UC-MSCs to inhibit T-cell proliferation in a IFNy-independent manner and the increase of this inhibition after pro-inflammatory stimulation (Mebarki et al, Stem Cell Res Ther. 2021 Nov 13; 12(1 ):571 .). In this study, it was confirmed that in the basal state (untreated), UC- MSCs from each single donor as well as from the pool, are able to suppress T-cells expansion in vitro in a dose-dependent manner (Figure 6A). The mean of this T-cells suppression was > 50% in each single donor and in the pool when UC-MSCs: PBMC ratio was 1 :10 and decreased significantly when the ratio UC-MSCs: PBMC was 1 :100 for UC-MSCsp001and 1 :300 for UC-MSCshighand UC-MSCsl0W. However, this activity was donor-dependent given that with the ratio UC-MSCs: PBMC = 1 : 10, the suppression of T cells proliferation was more important in UC-MSCshighthan in UC- MSCs1™' (82.9±12.1 % vs 51.1±15. 1 %, p=0.0278) before treatment (in the basal state) as well as after priming with IFNy (81 ,9±8.2% vs 58.0±10.8%, p=0.0204) and, tends to be higher with IFNy+TNFa (85.3±1.2% vs 46.8±28.0%, p>0.05) (Figure 5B). After priming with IFNy, T-cell suppression by UC-MSCshighand UC-MSCsp°01became similar at a ratio of 1 / 10, and were both significantly higher than UC- MSCsmediumand UC-MSCsl0W(Figures 6.D-F). Interestingly, UC-MSCsp°o1approximates the potential of UC-MSCshigh. After IFNy treatment, the inhibition of T- cells expansion by UC-MSCsp°o1was higher than UC-MSCslow(89.7±5.4% vs 58.0±10.8%, p=0.0062, Figure 5B) and than the theoretical average of the three donors (89.7±5.4% vs 67.1 ±16.4%, p=0.0053, Figure 5C). In other experiments, after treatment with IFNy+TNFa, the inhibition of T-cell expansion by UC-MSCsp°o1at a ratio of 1 / 10 was still significantly higher than UC-MSCsl0W(88.4±10.2% vs 60.5±7.6%, p=0.0209). Interestingly, the inhibition of T-cell expansion by UC- MSCspoolwas significantly higher than the theoretical average of individual donors at a ratio of 1 / 10 after treatment with IFNy (91.2±5.4% vs 64.3±16.9%, p=0.0004) and IFNy+TNFa (88.4±10.2% vs 64.5±15.8%, p=0.026) (Figures 6.G-I).Taken together, these results suggest that pro-inflammatory stimulation does not decrease the donor-dependent variability whereas the use of pooled UC-MSCs improves low immunomodulatory functions.8. Pooling UC-MSCs from high and low donors with decreased ratio ofUC-MSCshighis sufficient to increase the lowest immunoregulatory functionsIn order to optimize the constitution of pools, and facilitate pharmaceutical manufacturing, which is time and cost consuming, the immunomodulatory properties of UC-MSCs pooled from two donors, high and low, named UC-MSCsp°o1 2Dwas assessed. Regarding the clinical value (due to rarity) of UC-MSCshi0h(20% in this study), dose de-escalation of these cells in this 2D pool was performed. Thus, UC- MSCs00012Dconsisted of UC-MSCshi0hand UC-MSCsl0Wat ratio 1 :1 ; 1 :2 and 1 :5 (UC- MSCspool 2D 1 :1; UC-MSCspool 2D 1 :2; UC-MSCspool 2D 1 :5respectively). In the basal state, IDO was not expressed (<2%) in UC-MSCsp°01 2Dregardless of the ratio. After treatment with IFNy, IDO expression was significantly increased in UC-MSCspool 2Dat ratio 1 : 1 compared with UC-MSCsl0W(67.1 ±14.5% versus 10.1±8.7%, p<0.0001) and remained higher for UC-MSCspool 2Dat ratio 1 :2 (47.5±26.9%; p=0.0113) and in UC-MSCsp°01 2Dat ratio1 :5 (47.0±23.1 %; p=0.0125) (Figure 7A). Priming with IFNy+TNFa increased the difference between UC-MSCspool 2Dregardless of the ratio and UC-MSCsl0Wwhile IDO expression in UC-MSCspool 2Dreached that observed in UC-MSCshigh(83.5±9.0%) at ratio 1 :1 (81.4±8.5%) and remained high at ratio 1 :2 (71.9±14.4%) and 1 :5 (65.0±19.6%). Importantly, IDO expression in UC-MSCs pooled from two donors even at low ratio (1 :2 and 1 :5), was similar with the three donors pool (Figure 7B).To confirm these results, an MLR assay was performed using the pool 2D at ratios 1 :1 and 1 :2 (UC-MSCspool 2D 1 :1and UC-MSCspool 2D 1 :2). In the basal state, the suppression of T-cells expansion by UC-MSCspool 2D 1 :1tended to be better than UC- MSCs10” at a UC-MSCs / PBMC ratio = 1 / 10 (63.6±27.1% versus 51.1±15.1 %, p=0.8777) and became significantly higher at a ratio of 1 / 100 (68.5±7.1 % versus 18.7±20.4%, p=0.0133, Figures 7C-E). This suppression remained higher with UC- MSCs00012D 1 :2compared with UC-MSCsl0W(86.4±8.1 % versus 51 . 1 ±15.1 %, p=0.0194 and 83.3±10.8% versus 36.0±14.1%, p=0.0094 at an UC-MSCs / PBMC ratio = 1 / 10 and 1 / 30 respectively). These results highlighted that pooling is still effective even with a decreased dose of UC-MSCshigh. After priming with IFNy, T-cell suppression by UC-MSCsp°o1 2D 1 :2was still higher than UC-MSCsl0W(83.1±2.7% versus58.0±10.8%, p=0.0176 at an UC-MSCs / PBMC ratio = 1 / 10). Interestingly, the capacity of UC-MSCsp°o1 2Dto suppress T cells was at least similar or even better than the theoretical mean of the two individual high and low donors (Figure 7F-H). Finally, the comparison of the 3D pool with the 2D pools at ratios of 1 :1 and 1:2 suggested a similar capacity to suppress T-cell expansion or even a higher T-cell suppression with the pool 2D 1 :1 (68.5±7.1% versus 30.7±4.5%, p=0.0422 in the basal state and with ratio UC-MSCs / PBMC = 1 / 100, Figure 7I-K)Example 3. DiscussionMSCs derived from fetal tissues and in particular from the Wharton’s jelly of UCs, are emerging as a promising perspective to develop cellular immunotherapies for the treatment of immune and inflammatory diseases. Compared with MSCs derived from adult tissues, UC-MSCs are highly proliferative and immunosuppressive due to their primitive status. However, the donor-dependent heterogeneity of UC-MSCs is greater than that of MSCs derived from adult tissues, which is a real challenge to develop standardized UC-MSC-based medicinal products and to predict the clinical response. Several studies have suggested that the baby’s gender could be a variability factor of the biological properties of the MSCs while others suggest that the donor’s age may have an impact. In the present study, no differences in IDO expression or suppression of T-cell proliferation were observed according to the age of the mother, the term of delivery, the gender and weight of the baby, or the weight of the UCs. This can be explained by the primitive nature of UC-MSCs compared with MSCs derived from adult tissues. Therefore, the clinical characteristics of mothers or babies cannot be used as predictive factors.Several strategies have been used to reduce this biological variability and develop MSC-based medicinal products with reproducible immunomodulatory functions. Zhang et al. (Cell Death Dis. 2021 Apr 6;12(4):357), indicated that interdonor variabilities of UC-MSCs immunosuppressive properties can be eliminated after treatment with two pro-inflammatory cytokines, IFNy and TN Fa. However, in the present study, the persistence of variability in immunoregulatory properties between donors after treatment with IFNy and IFNy+TNFa is demonstrated. Indeed, high IDO expression (>60%) was only found in two thirds of the donors, while one third had low IDO expression (<30%). The MLR potency assay corroborated this heterogeneity, with the persistence of three distinct profiles of immunomodulatory potential, even after pro-inflammatory stimulation. Thus, the results of example 2demonstrated that treatment with cytokines is insufficient to eradicate the heterogeneity and enhance the lowest immunomodulatory properties of UC-MSCs. In addition, because it has been shown to reduce MSC proliferation, treatment with cytokines during clinical manufacturing should be avoided (Zhang et al., 2021 , op. cit.).The production of the large quantities of cells needed to develop MSC-derived medicinal products for clinical use, requires extensive ex vivo expansion over several passages. This strategy may introduce the risk of genetic instability and chromosomal abnormalities as well as an alteration of the biological properties of the MSCs. Pooling MSCs from multiple donors is emerging as a promising strategy to reduce the donor-to-donor heterogeneity and to produce an extended number of standardized clinical batches. Due to their low immunogenicity and long-term demonstrated safety, MSCs can be used to produce pooled off-the-shelf medical products. A non-clinical study performed in rats and rabbits showed the absence of acute or chronic toxicity, tumorigenicity, and teratogenicity following administration of human bone marrow-MSCs pooled from three healthy donors in an equal proportion (Rengasamy et al., Indian J Med Res. 2016 Dec; 144 (6): 852-64). Another study in immunocompetent Balb / c mice, showed that bone marrow-derived MSCs were rejected faster than UC-MSCs, suggesting that UC-MSCs are more tolerogenic. In the present study, it is showed that the induction of HLA-DR expression by pro-inflammatory treatment is donor-dependent. Pooling normalized HLA-DR expression without increasing the expression of HLA-DR or the costimulatory molecules (CD40, CD86) compared with UC-MSCshigh, demonstrating the possibility of using a pooled allogeneic product without altering the tolerogenic profile of UC-MSCs. In addition, the batch-to-batch variability of pooled MSCs is limited compared with batches produced with MSCs from a single donor, providing an opportunity to standardize medicinal products. Despite several advantages, this strategy has shown limited benefits when MSCs from randomly selected donors were pooled (Hejretova et al., Cell Tissue Bank. 2020 Mar;21 (1):119-29).In this study, the profile of donors was graded based on the immunoregulatory properties of the UC-MSCs, in order to select donors whose cells were to be included in pools. The aim was to prepare pools including UC-MSCs isolated from at least one donor with a high immunomodulatory potential.First, the donor-to-donor heterogeneity of the UC-MSCs immunoregulatory functions was confirmed. The expression of IDO was increased after priming cellswith IFNy and IFNy+TNFa, but in a donor-dependent manner. The suppression of T-cells proliferation by UC-MSCs confirmed donor-dependent differences in their immunoregulatory properties in the basal state (without pro-inflammatory treatment). The results demonstrated that the immunosuppressive ability of UC-MSCs is innate, regardless of the donor, as shown in previous study (Mebarki et al., Stem Cell Res Then 2021 Nov 13; 12(1 ):571 ), in contrast to adult tissues-derived MSCs which require pro-inflammatory induction (Ren et al., Cell Stem Cell. 2008 Feb 7;2(2): 141- 50).Based on the capacity of UC-MSCs to suppress T-cell proliferation in the basal state, the UC donors were classified into three profiles. Donors were graded as low if the suppression of T-cell proliferation was below the median, as medium if above the median, and as high if the results were in the last quartile. Consequently, five donors were graded as low, three as median, and two as high.To assess the benefits of pooled UC-MSCs compared with single donors, cells isolated from one donor of each profile were pooled in an equal proportion. First, the results showed that pooled UC-MSCs displayed a similar morphology and phenotype to cells from individual donors. IDO expression was significantly different between the three donor profiles, with the highest expression by UC-MSCshighafter treatment with IFNy and IFNy+TNFa, despite a similar IFNy-receptor expression. Indeed, the results demonstrated that the expression of IFNy-receptor is donorindependent and is not associated with the priming of MSCs by IFNy as previously shown (Chan et al., Blood. 2006 Jun 15;107(12):4817-24). Interestingly, pooling UC-MSCs (UC-MSCsp001) increased the lowest expression of IDO (UC-MSCsl0W), while the expression of the IFNy receptor by UC-MSCsp°o1remained unchanged. In addition, IDO expression by UC-MSCsp°o1was similar to the theoretical mean, highlighting that pooling the cells resulted in homogenization. These results were confirmed by the MLR assay which showed persistence of donor-dependent variability. In the basal state (ratio 1 :10), the suppression of T-cell proliferation by UC-MSCshighwas higher than UC-MSCsmedium(p>0.05) and UC-MSCslow(p=0.0278) whereas UC-MSCsp°o1tends to increase T-cells suppression compared to UC- MSCs1™' (Figure 6A). In the presence of IFNy (ratio 1 :10), T-cells suppression by UC- MSCspoolwas significantly higher than UC-MSCslow, UC-MSCsmediumas well as the theoretical mean of single donors. Similar results were found after priming cells with IFNy+TNFa. Taken together, the results showed that pooling UC-MSCs not only reduces donor-dependent heterogeneity, but also increases the lowestimmunomodulatory functions, and this capacity is increased in a pro-inflammatory environment. Moreover, pooling UC-MSCs derived from a high donor profile with a low one at a ratio of 1 :2 was sufficient to enhance the lowest immunomodulatory properties. These findings offer new perspectives for clinical applications. The use of UC-MSCs from one donor graded as high in different pools will offer the possibility of increasing the number of clinical batches, and hence treating more patients with a higher expectation of immunoregulatory effects. This is of interest as only a few donors are qualified as high (20% in the present study). This is considered the first study demonstrating such results. Indeed, previous studies have pooled MSCs isolated from randomly selected donors without grading their biological properties. Kannan et al., pooled UC-MSCs based on the gender of the baby’s without preselection. In a more recent study, the same team assessed the biological properties of several pools of bone marrow-derived MSCs, prepared from three randomly selected male donors. They suggested an improved immunosuppressive capacity of pooled batches due to synergistic effects between single donors, although this remains debatable as analysis was performed on expanded cells until P5, with unequal proportions between individual donors.In the study, UC-MSCs with greater expression of IDO were shown to be able to largely inhibit T-cell expansion. Other mediators such as prostaglandin E2 (PGE2), TGF-P, TSG-6 and NO have also been described, underlining the role of the paracrine effect of MSCs to regulate immune cells. Despite the growing interest in paracrine mechanisms, contact-mediated immunomodulatory effects are critical to guarantee the full efficacy of MSCs immunosuppressive functions. Ren et al., showed that MSCs failed to suppress T-cell expansion when they were separated by a permeable membrane, highlighting the importance of contact-dependent mechanisms (Ren et al., 2008, op. cit.). The suppression of monocyte differentiation into dendritic cells, the switch of macrophages into an anti-inflammatory phenotype, the differentiation of CD4+T cells into T regulatory cells and the inhibition of Th17 cells are mediated by cell-to-cell interactions via cell-surface molecules. It was therefore decided to analyze the expression of immune and adhesion surface markers involved in cell-to-cell contact. First, the results showed a high heterogeneity in surface markers expression depending on the donor. CD200 was high in UC-MSCshigh, ALCAM (CD166), CD146 and VCAM-1 (CD106) in UC- MSCsmedium, whereas PDL-1 presented a similar expression in UC-MSCshighand UC- MSCs10”. Interestingly, despite these variations, pooling UC-MSCs resulted in anincreased expression of all these molecules. ALCAM (CD166), CD146 and CD200 were increased in UC-MSCsp°o1in the basal state and after priming, whereas VCAM- 1 (CD106) and PDL-1 needed a pro-inflammatory stimulation even after pooling MSCs. ICAM-1 (CD54) was significantly higher in UC-MSCshighat basal state only and was enhanced by IFNy or IFNy+TNFa to reach around 100% in all donors and in the pool.Finally, CD44 was homogeneously expressed regardless of the donor and culture conditions. These results suggest that the immunomodulatory functions of UC-MSCs could be increased by pooling cells without pro-inflammatory priming. The immunoregulatory mechanism of pooled cells appears to involve cell-cell interactions through the induction of the expression of adhesion molecules ALCAM, CD 146 and CD200. CD200 expressed on the surface of MSCs has been described to bind to its receptor CD200R expressed on macrophages, leading to their switch into anti-inflammatory phenotype. In the study, the expression of VCAM-1 (CD106) and PDL-1 was higher in pooled cells after inflammatory priming, although not in all single donors. ICAM-1 and VCAM-1 play a key role in the contact-mediated immunoregulation by MSCs, and PD-L1 is involved in the inhibition of T and B-cell activation in a cell-contact manner.Taken together, the results demonstrated that pooling UC-MSCs decreases donor-dependent heterogeneity and that the inclusion of UC-MSCshighin pools enhances the lowest immunoregulatory properties. In addition, pooling only two donors, including one donor graded as high and another as low, appears to be sufficient to improve UC-MSCs immunoregulatory capacities. These results were confirmed even when the quantity of UC-MSCsl0Wwas twice that of UC-MSCshigh.In this study, the donor-dependent heterogeneity of UC-MSCs immunomodulatory functions was confirmed. Regarding these properties, donors were graded into three profiles as high, medium, and low, and then prepared pools containing UC-MSCs with high immunomodulatory properties. The results demonstrated that pooling cells with at least one high donor, decreases the donor- to-donor variability and improves the lowest immunomodulatory functions, without increasing the immunogenicity of the product. This strategy offers a new perspective to produce large scale standardized UC-MSC-based medicinal products, with an improvement in their immunomodulatory properties for the treatment of severe and / or refractory immune or inflammatory diseases.

Claims

CLAIMS1. A method for grading the immunomodulatory property of human mesenchymal stromal cells (MSCs), comprising a. Revealing immunomodulatory properties of mesenchymal stromal cells cultured in a resting state and / or primed in pro-inflammatory conditions, wherein the immunomodulating property of the cells is the ability to inhibit lymphocyte proliferation, as measured by Mixed Lymphocyte Reaction b. Obtaining a numerical value associated with the immunomodulatory property of the cells c. Grading the immunomodulatory property of the population of cells as high, medium or low, wherein a grade “high” or “medium” is assigned when the numerical value associated with the immunomodulatory property of the cells is above a median value, and wherein a grade “low” is assigned when the numerical value associated with the immunomodulatory property of the cells is below the median value, wherein the median value is the median value from a series of numerical values associated with the immunomodulatory properties, measured from multiple populations of mesenchymal stromal cells from multiple donors.

2. The method of claim 1 , wherein the grade “high” is assigned when the numerical value associated with the immunomodulatory property of the cells is within the last quartile, and wherein the grade “medium” is assigned when the numerical value associated with the immunomodulatory property of the cells is within the third quartile of the series of numerical values associated with the immunomodulatory properties of multiple populations of mesenchymal stromal cells from multiple donors.

3. The method of claim 1 , wherein the grade “high” is assigned when the numerical value associated with the immunomodulatory property of the cells is above the sum of median and interquartile, and wherein the grade “medium” is assigned when the numerical value associated with the immunomodulatory property of the cells is above the median and below sum of median and interquartile of the seriesof numerical values associated with the immunomodulatory properties of multiple populations of mesenchymal stromal cells from multiple donors.

4. The method of any one of claims 1 to 4, wherein the median value is calculated for values associated with mesenchymal stromal cells from 10 or more donors.

5. The method of any one of claims 1 to 5, wherein the mesenchymal stromal cells are umbilical cord derived mesenchymal stromal cells.

6. A method for preparing a pool of mesenchymal stromal cells, comprising: a. Providing mesenchymal stromal cells from at least two donors b. Testing the mesenchymal stromal cells of each donor, with regards to their immunomodulatory properties c. Selecting first mesenchymal stromal cells from a first donor, wherein the cells present high immunomodulatory properties d. Selecting second mesenchymal stromal cells from a second donor, wherein the cells present medium or low immunomodulatory properties e. Mixing the first and second selected mesenchymal stromal cells to obtain a pool of mesenchymal stromal cells.

7. The method of claim 6, wherein testing and grading of the mesenchymal stromal cells is performed by the method of any of claims 1 to 5.

8. The method of claim 6 or 7, further comprising preparing multiple aliquots of the cells of the pool.

9. The method of any one of claims 6 to 8, further comprising freezing the pool or the cells from individual donors.

10. A pool of mesenchymal stromal cells, wherein the pool contains cells from at least two donors, wherein the cells of one donor are graded as having high immunomodulatory property, and wherein the cells of the other donor(s) are graded as having medium or low immunomodulatory property11 . The pool of claim 10, for use thereof as a medicament.

12. A pool of mesenchymal stromal cells, wherein the pool contains cells from at least two donors, wherein the cells of one donor are graded as having high immunomodulatory property, and wherein the cells of the other donor(s) are graded as having medium or low immunomodulatory property, for use thereof for the treatment of immune or inflammatory diseases.

13. The pool of mesenchymal stromal cells for use according to claim 12, wherein the cells are provided at a dose comprised between 105 / kg to 107 / kg to the patient, in one or more administrations.

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