EP3818145A1

EP3818145A1 - Neonatal stromal cells having low mhc-i expression and uses thereof

Info

Publication number: EP3818145A1
Application number: EP19756214.3A
Authority: EP
Inventors: Rodolphe RAKIC; Nathalie SAULNIER; Stéphane MADDENS
Original assignee: Vetbiobank
Current assignee: Vetbiobank
Priority date: 2018-07-02
Filing date: 2019-07-02
Publication date: 2021-05-12
Also published as: WO2020008139A1; US20210269768A1

Abstract

The invention relates to a population of neonatal stromal cells (NSC) showing low MHC-I (MHC-Ilow) expression and optionally high CD90 expression, a pharmaceutical composition and an injectable solution comprising said NSC population, as well as a method for obtaining said composition from neonatal tissue. The invention also relates to the advantageous use of said cell population in regenerative, veterinary or human medicine, more specifically in the treatment of osteoarthritis and chronic inflammatory diseases and more generally for treating tissue lesions, degenerative diseases, autoimmune diseases, infectious diseases with or without an inflammatory component, or for treating graft rejection and tumour diseases.

Description

NEONATAL STROMAL CELLS WITH LOW EXPRESSION OF MHC-I AND USES THEREOF

Technical area

The invention relates to a population of neonatal stromal cells (CSN), which can be industrialized, weakly expressing the major histocompatibility type I complex (called CMH-I low or CMH-I ^L ) and optionally strongly CD90 (called CD90 high or CD90 ^h ), a composition comprising said population of CSN CMH-I ^L and optionally CD90 ^H and the process for obtaining this composition from neonatal tissues. In particular, the invention relates to the advantageous use of this population of cells in regenerative, veterinary or human medicine, more specifically as a treatment for osteoarthritis and chronic inflammatory diseases and more generally for the treatment of tissue damage, degenerative diseases , autoimmune diseases, infectious diseases with or without an inflammatory component or in transplant rejection and tumor diseases. The invention also relates to a ready-to-use pharmaceutical composition comprising such a population of CSNs.

Context

Mesenchymal stromal cells (MSCs) were first isolated from the bone marrow; however, their frequency is quite low in this tissue and declines with age (0.01% of total mononuclear cells in adults) (Bruder et al., 1997). Cells with similar characteristics were subsequently identified in the stromal vascular fraction of adipose tissue in a larger proportion. MSCs are characterized by the expression of a panel of surface markers CD105, CD73, CD90 and do not express CD45, CD34, CD14 or CD1 lb, CD79alpha or CD19 and HLA-DR (Dominici, 2006). Furthermore, MSCs must be able to differentiate in adipogenic, osteogenic, chondrogenic lineages in vitro, using suitable differentiation media. In the context of therapeutic use, MSCs are usually isolated from adult tissue (bone marrow, adipose tissue) taken from a patient, and reinjected into the patient himself following an amplification phase in the laboratory. A delay is therefore necessary to administer the therapy to the patient.

MSC populations isolated in a conventional manner from several samples from the same tissue source but from different subjects, are heterogeneous with each other (inter-population heterogeneity) from the point of view of markers cell. In addition, it is also considered that within a cell population originating from the same sample, several MSC phenotypes coexist (intra-population heterogeneity). This implies a lack of reproducibility in the context of their industrialization and clinical efficacy (Phinney, 2012). Indeed, the cell phenotype influences biological properties and characteristics. This inter- and intra-population heterogeneity is notably observed for the CMH-I and CD90 membrane markers present in MSCs. Several studies show variations in the expression of these markers according to tissue sources, isolation or amplification methods.

Jacobs et al. (2013) mention multipotent human adult progenitor cells isolated from bone marrow, having a low expression of MHC-I and a high expression of CD90, capable in particular of differentiating into osteoblasts and chondrocytes.

Tessier et al, (2015) and Davies et al, (W02004 / 072273) described neonatal MSCs weakly expressing MHC-I and expressing CD9G, however the work of these teams shows both a strong fluctuation in expression of MHC-I among the strains isolated during the cryopreservation and amplification process (Lepage et al, 2019) (Sarugaser, et al, 2005). Portmann-Lanz et al, (2006) described MSCs isolated from fetal membranes and human placental tissues and show significant variability in the expression of CD90 and MHC-I in these MSCs.

These different works descriptively exploit the expression of MHC-I without identifying the effects of these differences of expression on the biological properties of MSCs. Taking into account the fluctuation of these two markers, CMH-I and CD90, and the associated functional characteristics of MSCs were not investigated.

Certain tissue sources, such as the placenta, show strong differential expression of these markers, thus limiting the reproducibility of an industrialization and clinical application strategy.

Furthermore, it should be noted that an important technical constraint lies in the methods of analysis of expression of CMH-I and / or of CD90 in these studies. Indeed, all of these works define their cells as being positive or negative for these markers. This notion of positivity or negativity requires the determination of a threshold whose relevance is dependent on many technical and biological parameters. it implies, among other things, analysis limits for heterogeneous MSC populations and / or for weakly expressed markers.

None of the characteristics of the cells described in these documents makes it possible to envisage a large-scale reproducible culture and therefore an industrialization of cell production, in particular for a therapeutic use of these cells.

Goals

One objective is to provide a population of cells intended for cell therapy, having a good proliferation capacity to allow its production on an industrial scale and in a reproducible manner. Another objective is to provide a population of cells intended for cell therapy, possessing capacities to differentiate into other cell types in particular into chondrocytes while limiting the risk of ectopic tissue formation. Another objective is to provide cells capable of interacting with cells of the immune system and regulating their activity. Another objective is to provide new means of effective treatment of tissue damage, degenerative diseases, autoimmune diseases, inflammatory diseases, infectious diseases or even in transplant rejection and tumor diseases, especially in non-human mammals, especially dogs, horses or cats, or in humans.

Yet another objective consists in providing a process for obtaining the cell population meeting the criteria sought, from samples taken in a non-invasive manner to allow industrialization of the production and this in a reproducible manner.

The inventors have surprisingly discovered that the neonatal tissues comprise a subpopulation of CSN of phenotype CMH-I ^L and optionally CD90 ^H having a high proliferation potential and thus allowing their industrialization and their advantageous use in cell therapy in particular for the treatment tissue damage and degenerative diseases in veterinary or human medicine.

The use of these cells is all the more justified for the treatment of joint pathologies, in view of their high potential for chondrogenic differentiation, their low potential for osteogenic differentiation and their capacity for immunomodulation. On the other hand, the under expression of MHC-I among these cells could induce a response limited immune system vis-à-vis these cells, compared to that induced vis-à-vis cells expressing strongly MHC-I.

The inventors have also succeeded in overcoming the strong inter- and intra-population heterogeneity existing in the placenta to provide a homogeneous population of CMH-1 ^L placental CSNs and optionally CD90 ^H having a high proliferation potential and thus allowing their industrialization and their use. advantageous in cell therapy.

In addition, the biological properties of the CSNs isolated by the inventors remain stable during the stages of cryopreservation / thawing.

Detailed description CNS population

A first object of the present disclosure relates to a population of neonatal stromal cells (CSN), comprising neonatal stromal cells of phenotype CMH-I ^L , and optionally of phenotype CD90 ^H. The inventors have discovered that these cells, isolated from a neonatal biological sample, have a high multiplication capacity, thus giving them a high industrialization potential and a high therapeutic potential.

CMH-I corresponds to the major class 1 histocompatibility complex, expressed almost ubiquitously within the body.

MHC-I, also known as HLA-I ("human leukocyte antigen class I") in humans, is derived from the expression of a family of genes called HLA genes. Among these genes we can cite HLA-A, HLA-B and HLA-C which code for the classic forms of HLA-I. In dogs, MHC is also called DLA (Dog Leukocyte Antigen), in horses, we speak of ELA (Equine Leukocyte Antigen), and in cats, we speak of FLA (Feline Leukocyte Antigen).

CD90 corresponds to the membrane protein "Thy-l Cell Surface Antigen" and is expressed by a large majority of stroma cells. By "MHC-I ^L phenotype" is meant that the CSNs have very low expression, or an absence of expression of MHC-I. By "CD90 ^H phenotype" is meant that the CSNs have a strong expression of the CD90 surface antigen.

The cells according to the present disclosure are naturally of weak MHC-I phenotype (MHC-I ¹ ) and optionally strong CD90 (CD90 ^H ), that is to say that the weak expression of MHC-I and the strong expression CD90 is not a phenotype resulting from genetic modification in the cell population.

In a particular embodiment, the population of CSN comprises neonatal stromal cells of phenotype CMH-I ^L and phenotype CD90 ^H (denoted CMH-I ^L / CD90 ^h ).

The expression of these two markers in a cell population can be assessed by the flow cytometry technique.

A cell population can be described as homogeneous when a population of CSN (isolated from a single given source) is homogeneous from the point of view of CMH-I and CD90 expression (absence of intra-population heterogeneity ), that is to say that the cells all have a similar level of expression of MHC-I and of CD90, and therefore all exhibit the same phenotype with respect to the expression of markers MHC-I and CD90 (Figure 12). A cell population is described as heterogeneous, when a CSN population (isolated from a single given source) is made up of different sub-populations expressing CMH-I and / or CD90 differently. We are talking about intra-population heterogeneity.

In the case of a homogeneous population, the analysis of the expression of the markers CMH-I and CD90 can be carried out by flow cytometry by a strategy known as simple labeling, that is to say that the labeling of each markers are produced independently in 2 separate tubes.

In the case of a heterogeneous population, the analysis of the expression of the markers CMH-I and CD90 can be carried out by flow cytometry by a double marking, that is to say a simultaneous marking of the two markers in a single tube for the same cell population in order to properly discriminate between sub-populations. Double labeling can also be used in the case of a homogeneous population. When considering a population of CSN comprising cells of phenotype of interest MHC-I ^L / CD90 ^h , said population is considered to be heterogeneous when said population of CSN comprises between 10% and 90% of cells of phenotype MHC-I ^L / CD90 ^H

When considering a population of CSN comprising cells of phenotype of interest MHC-I ^L / CD90 ^h , said population is considered to be homogeneous when said population of CSN comprises more than 90% of cells of phenotype MHC-I ^L / CD90 ^H OR less than 10% of cells of phenotype CMH-I ^H / CD90 ^l .

The level of expression of a marker (weak or strong or lack of expression) can be assessed by a flow cytometry analysis technique correctly developed by a person skilled in the art. The notion of weak or strong expression implies a notion of discrimination threshold making it possible to correctly distinguish this population from populations which, conversely, have strong or weak expression of said marker from an analytical point of view. The methodology of marker expression analysis is dependent on the marker labeling strategy used. For example, using fluorochrome with different fluorescence yields will not have the same detection ability. The use of labeling strategies with signal amplification systems (biotin-avidin for example) will also influence the detection capabilities of the antibody of interest. It is therefore imperative to define adequate positivity / negativity thresholds according to the strategy used.

MHC-I expression in particular in CSNs is relatively low compared to other cell types such as fibroblasts or peripheral blood mononuclear cells (PBMC). The inventors have therefore developed a labeling strategy for MHC-1 sensitive enough to detect small differences in expression of this marker in CSNs.

The results of Figures 12 and 14 show that the analysis of MHC-I and CD90 by simple labeling can be limiting in the case of intra-population heterogeneity. Analysis of fluorescence means for heterogeneous populations does not allow it to be established with precision whether a total population is of phenotype CMH-I ^L / CD90 ^H OR CMH-I ^H / CD90 ^l . The double labeling methodology thus provides better resolution in the context of the characterization of CSN populations showing strong intra-population heterogeneity. Double marking of CMH- and CD90 markers

An example of simultaneous labeling (also called double-labeling or immunophenotypic co-labeling) of MHC-I and of CD90 within the same population of CSN, can be carried out using:

- for the labeling of CMH-I, a primary mouse anti-MHC-I IgG2a antibody, such as the primary anti-MHC-I antibody DG-BOV2001 / DG-H58A IgG2a (Monoclonal Antibody Center Washington State University), revealed with a secondary goat antibody F (ab ') 2 anti-secondary mouse IgG coupled to allophycocyanin (APC), and

- for labeling CD90, an anti-CD90 monoclonal antibody coupled to phycoerythrin (PE), such as the anti-CD90 / Thyl Antibody YKIX337.217 rat monoclonal antibody (PE).

After labeling, the fluorescence of the APC and PE fluorochromes is analyzed. In order to discern the CSN subpopulations, a 2D representation of the APC and PE fluorescence is performed. The results are compared with those obtained with the use of the isotypes coupled to the respective fluorochromes described above.

A population or subpopulation of CSN is qualified as MHC-I ^L / CD90 ^H if the ratio of the mean fluorescence intensity (MFI) between the MHC-I and its control isotype (also called relative MFI or rMFI) is less than a threshold of 20, more particularly 15, more particularly 10 and if the rMFI between the marker CD90 and its control isotype is greater than 15, more particularly 20.

A population or subpopulation of CSN is qualified MHC-I ^H / CD90 ^L if the ratio of the means of fluorescence intensity (MFI) between the MHC-I and its control isotype is greater than 20, more particularly 15, more particularly 10 and if the rMFI between the CD90 marker and its control isotype is less than 15, more particularly 20.

With double labeling, the presence of different CSN subpopulation profiles with regard to the expression of the markers CMH-I / CD90 (CMH-I ^L , CMH-I ^H and CD90 ^H , CD90 ^L ) is highlighted. evidence, within the same cell population from the same sample (Figure 12). These different phenotypes in the expression of MHC- markers I / CD90 imply biological differences between the different subpopulations highlighted.

An analysis of the expression of the two markers CMH-I and CD90 can be carried out for several populations of CSN from sources defined and isolated according to the same experimental protocol. The inventors confirmed by their method the existence of an inter-population heterogeneity with regard to the expression of these two markers among the populations of CSN coming from neonatal tissues of different natures (placenta, umbilical cord, blood of umbilical cord) (Figure 13).

Simple marking of CMH- and CD90 markers

Alternatively, a simple labeling of CMH-I and CD90 can be carried out.

An example of simple labeling of CMH-I within a population of CSNs, for a cytometric analysis, can be carried out using the primary anti-CMH-I antibody DG-BOV2001 / DG-H58A IgG2a (Monoclonal Antibody Center Washington State University) and the primary isotype control Mouse-COL2002 / COLIS205C IgG2a (Monoclonal Antibody Center Washington State University). These antibodies not coupled to a fluorochrome are used in conjunction with the use of a rabbit F (ab ') 2 anti-mouse IgG STAR12A (AbSerotec) antibody labeled with phycoerythrin (PE). This method of cytometric analysis is described in more detail in Example B. 2).

In this embodiment of simple labeling, a population of CSN is considered to be of low MHC-I phenotype (MHC-I ^L ), if the ratio between the mean fluorescence intensity (MFI) values such that measured by flow cytometry, cells labeled with an antibody specific for an MHC-I epitope relative to the MFI values of cells labeled with the control isotype is less than 3, in particular less than 2.5, in particular less than 2, more particularly less than 1.96.

Conversely, a population of CSN is considered to have a strong MHC-1 phenotype (MHC-I ^h ), according to the same method, if the ratio between the MFI values, as measured by flow cytometry, of the cells labeled with a specific antibody of an MHC-1 epitope relative to the MFI values of the cells labeled with the control isotype greater than 1.96, in particular greater than 2, in particular greater than 2.5, more particularly greater than 3.

In another embodiment, the simple labeling of CMH-I within a population of CSNs, for a cytometric analysis, can be carried out using the primary anti-CMH-I antibody DG-BOV2001 / DG-H58A IgG2a (Monoclonal Antibody Center Washington State University) and the primary isotype control Mouse-COL2002 / COLIS205C IgG2a (Monoclonal Antibody Center Washington State University). These antibodies not coupled to a fluorochrome are used in conjunction with the use of a secondary goat F (ab ') 2 anti mouse secondary IgG (eBio science) antibody coupled to allophycocyanin (APC). This method of cytometric analysis is described in more detail in the part of Example B. 2).

In this embodiment of the simple labeling, a population of CSN is considered to be of phenotype CMH-I ^L , if the ratio between the values of mean fluorescence intensity (MFI, mean fluorescence intensity) as measured by flow cytometry , cells labeled with an antibody specific for an MHC-I epitope with respect to the MFI values of cells labeled with a control isotype G is less than 12, in particular less than 10, more particularly less than 9.

Conversely, a population of CSN is considered to be MHC-1 ^H , according to this method, if the ratio between the values of MFI, as measured by flow cytometry, of the cells labeled with an antibody specific for an epitope of MHC-1 by ratio of the MFI values of the cells labeled with the control isotype is greater than 9, in particular greater than 10, more particularly greater than 12.

In another embodiment of the simple labeling, a population of CSN is considered to be of phenotype CMH-I ^L , if the ratio between the values of mean fluorescence intensity (MFI, mean fluorescence intensity) as measured by cytometry of flow, cells labeled with an antibody specific for an MHC-I epitope relative to the MFI values of cells labeled with control isotype G is less than 20, in particular less than 15, more particularly less than 10.

The determination of these thresholds is explained in Example A, part 2) e) and in Figure 3. Conversely, a population of CSN is considered to be of MHC-I ^h phenotype, if the relationship between the intensity values of mean fluorescence (MFI, mean fluorescence intensity), as measured by flow cytometry, of cells labeled with an antibody specific for an MHC-I epitope with respect to the MFI values of the cells labeled with the control isotype is greater than 20, in particular greater than 15, more particularly greater than 10.

In addition to the method based on flow cytometry, the analysis of the MHC-I phenotype can also be confirmed at the protein and / or transcriptional level.

At the protein level, the expression of MHC-I, whatever the species of interest, can be studied by the use of antibodies or fragments of antibodies (monoclonal or polyclonal) specifically directed against one or more epitopes of HLA- A, B, C, or their counterparts depending on the species. The techniques associated with these antibodies include cytometric analysis, Westem-blot, ELISA test, immunofluorescence and / or immunohistochemistry, among others. More generally, technologies involving the interaction between MHC-I and a labeled protein, such as an inhibitor, can be used as analytical tools. Other technologies using probe-labeled oligonucleotides can be used to evaluate this expression such as the use of aptamers, RNA probes and / or DNA probes.

At the transcriptional level, the analysis of the expression of the various genes can be carried out by techniques of final time PCR, RTqPCR, digital PCR and / or RNA microarray. It is also possible to measure the transcriptional expression of a component of MHC molecules, as may for example be beta 2 microglobulin (B2M).

Concerning the simple labeling of CD90 for a flow cytometry analysis, this can for example be carried out using an anti-CD90 antibody coupled to fluorochrome PE, such as the CD90 antibody YKIX337.217 (Bio Rad) compared to the signal obtained with an isotypic control coupled to the same fluorochrome Mouse (BALB / c) IgGl, K MOPC-2l (AbSerotec). In this embodiment, a population of CSN is considered to be of strong CD90 phenotype or so-called strong CD90 (CD90 ^H ), if the ratio between the mean fluorescence intensity (MFI) values as measured in flow cytometry, cells labeled with an antibody specific for a CD90 epitope relative to the MFI values of cells labeled with the control isotype is greater than 15, more particularly greater than 20. (Example B part 2)). Conversely, a population of CSN is considered to be of CD90 ^L phenotype, if the ratio between the values of mean fluorescence intensity (MFI, mean fluorescence intensity) as measured by flow cytometry, cells labeled with a specific antibody d an MHC-I epitope relative to the MFI values of cells labeled with the control isotype is less than 15, more particularly less than 20. In addition, the method based on flow cytometry, the analysis of the CD90 phenotype can also be confirmed at the protein and / or transcriptional level using antibodies specifically directed against one or more epitopes of CD90 and / or at the transcriptional level by the analysis of the Thyl gene coding for CD90 by PCR techniques in final time, RT- qPCR, digital PCR and / or RNA microarray.

Determination of the threshold

Whether by simple or double labeling, the evaluation of the level of expression of CMH-I and CD90 requires the determination of a threshold. By “determination of the threshold” is meant the methodology which a person skilled in the art can apply in order to highlight all of the differential expressions of MHC-I and / or of CD90 among the populations of cells and more particularly CSN and define an acceptable relative expression limit. The purpose of this threshold is to exclude CSNs overexpressing CMH-I and / or under expressing CD90 and to include those under expressing CMH-I and / or over expressing CD90 in the industrialization process.

The majority of work on MSCs described in the prior art, exploits the expression of membrane markers through the notion of positivity or negativity of a cell for a given marker (either the absence or the presence of this marker ). These methodological thresholds are determined from the population of CSNs whose aspecific epitopes have been saturated and marked by isotypic antibodies not specifically targeting any epitope (isotypic population). The positivity / negativity thresholds are generally set between 2 and 3 standard deviations of the isotypic population whose statistical distribution is Gaussian in nature. That is to say a threshold placed so that 95.45% -99.73% of the isotypic population corresponds to the negative population of the cells labeled for the marker of interest. For highly protein markers specific to a particular cell type, this threshold plays a role in the interpretation of the data.

However, in the case of MHC-I and CD90, these two proteins are expressed by a large majority of stromal cells and are not specific to CSM or CSN. There is also a more or less significant fluctuation in expression between the different cell types, including among the MSCs and the CSNs. Thus, placing the positivity / negativity thresholds between 2 or 3 standard deviations of the isotypic population can induce a significant methodological bias for weak expressions, such as for the analysis of the CSN described in the present invention.

Thus, despite the technical possibility of assessing the positivity or negativity of cells for MHC-I and / or CD90, it is possible that the criterion of belonging to these categories does not represent a biological reality. Indeed, the CMH-I ^L and / or CD90 ^{L character} only reflects a weak expression of this marker by the cells and implies a relative positivity for all the cells (FIG. 1E and FIG. 14).

Ideally and as part of a labeling strategy for a given antibody panel and for the analysis of MHC-I and / or CD90, a precise threshold can be determined by the skilled person through analysis a statistically sufficient number, or representative sample, of cell population belonging to the two categories: under-expressing CMH-I and over-expressing CMH-I by way of example which, respectively, allow industrialization of CSNs or do not allow it .

The industrial character, which is coupled with the weak expression of CMH-I and optionally of the phenotype CMH-I ^L / CD90 ^H to distinguish the two populations of CSN, can be replaced by another biological character insofar as this the latter can be correlated with the expression of MHC-I and or of CD90 (eg the character of chondrogenic differentiation).

The obtaining, by a person skilled in the art, of this sufficiently large number of CSN populations, or representative sample, from a statistical point of view in the two categories of CSN, makes it possible to determine a limit of discrimination between the two populations . In the context of the invention, this discrimination limit results in the determination of a relative expression threshold for CMH-I and / or CD90 and more precisely in the context of cytometric analysis by an rMFI threshold . By "discrimination limit" is meant the threshold and the fluctuations of this threshold allowing unambiguously to discriminate CSNs over-expressing CMH-I or CD90 and under-expressing CMH-I or CD90. This discrimination limit must make it possible to validate the high expression for the MHC-I or the low expression for the CD90 of a non-industrializable CSN population with high sensitivity, or to guarantee a high interpretation as true positive. Furthermore, this limit of discrimination must make it possible to validate the low expression for the CMH-I or a strong expression for the CD90 of a population of CSN which can be industrialized with a high specificity, or to guarantee a weak interpretation as a false negative Inasmuch as the number of populations of CSN analyzed is sufficiently large, those skilled in the art can refine this discrimination limit by relying on the curve of the receptor efficiency function (ROC curve).

The analysis of CMH-I has several repercussions from an industrial point of view because its expression is on the one hand specific to tissue origin but also dependent on the microenvironment of CSN in vivo and / or in vitro.

From a technical point of view, this discrimination limit, or threshold, can be used on the one hand to exclude from industrialization non-industrializable cells and on the other hand:

• CSNs that have already been stimulated to an inflammatory environment in vivo or ex vivo;

• CSNs contaminated by a population of CSNs that do not meet the selection criteria;

• CSNs contaminated by a population of another cell type expressing high levels of MHC-I (for example, fibroblasts).

Concerning CD90, the expression of the latter is dependent on the state of differentiation of the cells and serves as an indicator for another biological slope than those indicated by the expression of MHC-I (Sibov et al., 2012). It is possible for those skilled in the art to establish a method of analysis similar to that described for CMH-I.

It is therefore advantageous to use an immunological labeling strategy which makes it possible to assess the expression panel of these two markers.

By the notion of “panel” we mean, in order of size, at least 2 categories of expressions observable in different cell types and phenotypes of CSN analyzed in our experiments for the MHC-I and for the CD90 (Table 1). Table 1: Categories of observable expressions of the CMH-I and CD90 markers in industrializable CSNs, non-industrializable CSNs, CSNs stimulated by IFN-g and Fibroblasts.

The discrimination limit may vary depending on the analysis technique used or the settings of the analysis tools used. For example, in the case of a fluorescence cytometric analysis, it is possible to increase the acquisition signal during the cytometric analysis and to vary the positivity / negativity thresholds by:

An increase in the sensitivity of the photomultipliers,

• the use of secondary antibodies,

• the use of primary and / or secondary polyclonal antibodies and / or cocktails of monoclonal antibodies and / or having a better titer for the epitope of interest,

• an antibody with a high protein / fluorochrome ratio,

· An antibody coupled to a fluorochrome with high emission energy.

Thus, in order to allow the selection of the populations of interest, those skilled in the art can finely determine the discrimination limit according to three following aspects: the given analysis strategy, the given marking strategy and / or the configuration of the given analysis tool. As part of the analysis strategy used by the inventors, the methodology is detailed in the material and method and in the figures associated with this document.

The present invention therefore relates to a population of CSNs comprising CSNs of phenotype CMH-I ^L , and optionally of phenotype CD90 ^H.

By “cell population” is meant a set of cells comprising one or more different cell types, for example a mixture of cells at different stages of differentiation, comprising cells having the same tissue origin or originating from different tissue origins. According to one embodiment, the population of CSN comes from a neonatal tissue sample, in particular from one or more placentas and / or from one or more umbilical cords, and / or from one or more amniotic membranes, or d a sample of neonatal fluid, in particular the blood of one or more umbilical cords, or the amniotic fluid of one or more amniotic fluids.

The advantage of neonatal tissue makes it possible to have a large number of CSN CMH-I ^L / CD90 ^H from a sample.

For this, the extra embryonic appendages (placenta, umbilical cord, amniotic membrane) are generally removed aseptically during cesareans or parturition by natural route in pregnant females, preferably at term. For example, as soon as the newborn is removed from the amniotic sac and placed in safety, the extra embryonic tissue is immediately transferred to a transport box containing for example a phosphate-buffered saline solution from Dulbecco to be transported to the laboratory. Umbilical cord blood can be collected by puncture from the umbilical vein, in particular using a needle connected to a blood collection bag or a tube or any other container.

In another example, the amniotic fluid can be recovered by puncture through the amniotic membrane, in particular using a needle connected to a blood collection bag or a tube or any other container.

Ratio of CSNs of interest in the cell population and origin of CSNs

In a particular embodiment, the population of CSN according to the invention is characterized in that less than 20% in number, in particular less than 15%, more particularly less than 10% of the cells of said population are CMH-I ^H and optionally characterized in that less than 20% in number, in particular less than 15%, more particularly less than 10% of the cells of said population are CD90 ^L.

In a particular embodiment, the population of CSN according to the invention is characterized in that it comprises at least 80% in number, in particular at least 85%, more particularly at least 90% of cells of phenotype MHC-I ^L / CD90 ^h .

The population of CSN according to the invention therefore corresponds either to a homogeneous population of CSN of phenotype CMH-I ^L / CD90 ^h , that is to say a population comprising between 90 and 100% of CSN of phenotype CMH-I ^L / CD90 ^h , that is to say a heterogeneous population comprising between 80 and 90% of CSN of phenotype CMH-I ^L / CD90 ^H (Figure 13).

In a particular embodiment, the CSNs come from a neonatal tissue sample, such as one or more placentas and / or one or more umbilical cords, or one or more amniotic membranes, or a sample of neonatal fluid such as for example the amniotic fluid from one or more amniotic sacs and in particular the blood from one or more umbilical cords.

In a particular embodiment, the population of neonatal stromal cells, comprising neonatal stromal cells of phenotype CMH-I ^L , and optionally of phenotype CD90 ^H , is a population of CSN originating from one or more placentas.

In a particular embodiment, said CSN population is a CSN population of placenta and comprises at least 80% in number, in particular at least 85%, more particularly at least 90% of cells of phenotype MHC-I ^L / CD90 ^h .

In a particular embodiment, the sample comes from neonatal mammalian tissues or fluids, and in particular from dogs, cats, horses or humans. In a more particular embodiment, the neonatal sample comes from the dog or the cat.

Other structural features of CSNs of interest

CSNs can also be characterized by the fact that less than 10% of the cells express one or more of the following surface markers: CD1 lb, CD14, CD31, CD34, CD45, or, HLA-DR, also called CMH-II.

The CSNs can also be characterized by the expression at the transcriptional level of growth factors, such as the vascular endothelium growth factor (VEGF), the growth factor (HGF), the keratinocyte growth factor (KGF), transforming growth factor type b (TGF-b). Biological characteristics of the CSNs according to the invention

In addition to their structural properties, the CSNs according to the present disclosure can be characterized by their biological characteristics.

In fact, the CSNs according to the invention can be characterized by their capacity for cell proliferation.

Thus, in a specific embodiment, the CSN population is characterized in that at least 80% of the cells of said CSN population have a capacity for consecutive cell doubling greater than 20 cumulative doublings.

By "cell proliferation capacity" is meant that a population of CSNs according to the invention is capable of doubling in number, more than 20 times in total.

To assess the number of doublings, at each cell passage, the cells are detached from their support, centrifuged and then counted, for example by the trypan blue exclusion technique using an electronic counter. The number of doublings at each cell passage is calculated according to the following formula: Nb of doublings = LOG (Nf / Ni) / LOG (2) (Nf: number of final cells and Ni: number of initial cells). The total number of cell doublings is equal to the sum of the number of doublings accumulated during each cell passage.

The cells according to the present disclosure can be industrialized, that is to say that their original source can supply a large quantity of cells of interest and make possible cellular amplification in vitro on an industrial scale. According to one embodiment, the CSNs have a proliferation capacity greater than 20 cell doublings cumulated during at least 4 cell passages.

Among different populations of CSN isolated from the same source and more particularly of canine placenta, the inventors have shown that there is a disparity of proliferation potential, making a part of these CSN populations difficult to industrialize (Ligure 15).

The inventors have completely unexpectedly identified that the MHC-I ^L / CD90 ^H phenotype of CSNs is linked to the proliferation capacity of cells, allowing them to double a greater number of times in vitro than CSNs of phenotype. CMH- I ^H / CD90 ^L (Ligure 4) This cell doubling capacity therefore makes it possible to reach a sufficient number of cells for the preparation of therapeutic preparations and thus industrialize the production of these cells for use in particular in veterinary or human medicine. Furthermore, this doubling capacity has a limit, thus minimizing a genetic drift of the cells and the emergence of a neoplastic phenotype. This characteristic is an additional argument for the use of these CSNs as a cell or tissue therapy product.

The neonatal stromal cell population can also be characterized in that at least 80% of the cells of said CSN population have:

an adhesion capacity on a plastic support; and or

a potential for chondrogenic differentiation, and / or

an immunomodulatory potential.

In a specific embodiment, the population of neonatal stromal cells is also characterized in that at least 80% of the cells of said CSN population have a limited osteogenic differentiation potential or do not have osteogenic differentiation potential.

By "adhesion capacity on plastic support" is meant that the CSN population is characterized by its adhesion property on plastic support.

By “chondrogenic differentiation potential”, it is meant that the CSNs have the capacity to be able to differentiate into chondrocytes. The expressions “chondrocyte differentiation” and “differentiation into chondrocytes” can also be used interchangeably.

This capacity for differentiation into chondrocytes can be assessed by the protein and / or transcriptional study of specific markers of cartilage such as Collagen type II (COL2A1), SOX-9 (SOX9), aggrecan (ACAN), cartilage Oligomeric Matrix Protein (COMP), Collagen type IX (COL9A1), collagen type XI (COL11A1), collagen type IIB (COL2B) after induction of chondrogenesis. On the other hand, viscoelastic properties of the extracellular matrix, expressed by differentiated CSNs, approximate the viscoelastic properties of cartilage. These viscoelastic properties can thus serve as a characteristic for the evaluation of chondrogenesis.

In a preferred culture mode, to induce chondrogenesis, the CSNs are detached from their support by trypsinization and used to form micromasses by gravitation in a drop of amplification medium such as for example DMEM (lg / L of glucose) supplemented with 10% S VF (vol: vol), 2mM glutamine and 0 to 20 ng / ml fibroblastic growth factor (FGF2-P). A CSN micromass then forms after 24 hours of incubation, at the base of the drop. This micromass is recovered and placed in the presence of a chondrocyte differentiation medium composed of DMEM with 4.5 g / L of glucose and added with TGF-b3 or a combination TGF-pi / BMP-2, for 7 to 28 days. At the end, an RNA or protein extraction is carried out in order to analyze the expression of specific markers of the chondrogenic lineage such as collagen type II, aggrecan, COMP, SOX9.

The CMH-I ^L / CD90 ^H CSNs have an increased chondrocyte differentiation potential compared to the CMH-I ^H / CD90 ^L CSNs with regard to the size of the micromasses generated (see FIGS. 5A, 5B, 5C and 5D) and / or the expression of chondrogenic markers such as COL2A1, SOX9, as well as with regard to the expression of a sulphated extracellular matrix composed of type II collagen. This relationship is in agreement with the advantageous therapeutic use of CSNs according to the present disclosure in the context of arthropathy insofar as they can participate in the repair of tissue of articular cartilage.

The inventors have thus demonstrated that the MHC-I ^L / CD90 ^H phenotype of the CSNs according to the present disclosure can be correlated with a significant capacity for chondrogenic differentiation of the cells in comparison with CSM type cells, derived from adipose tissue, d amnion or bone marrow, also having this chondrogenic capacity.

Thus, in a particular embodiment, the population of CSN CMH-I ^L / CD90 ^H according to the invention, is characterized in that at least 80% of the cells of said population of CSN have a potential for chondrogenic differentiation. In particular, this chondrogenic differentiation potential is appreciated in relation to the expression of the Col2al gene, and is at least 10 times greater compared to a population of CSN CMH-I ^H / CD90 ^l .

By "immunomodulatory potential" is meant the immunomodulating capacity of CSNs.

This potential can be characterized by the ability of CSNs to express a set of immunomodulatory factors such as PGE2, IL-6, IL-10, TGL-b, IDO, iNOS, HGL, KGL, CCL2 and / or TSG- 6. Indeed, in the presence of an inflammatory context, the CSNs can modify their phenotype. The inflammatory context can be mimicked in vitro by stimulation of the cells by means of cytokines and / or growth factors such as ILN-g, IL-1, IL- 6 and / or TNF-a. A modification of the CSN phenotype results in the ability of the CSNs to modify the transcriptional and / or protein expression of markers involved in immunomodulation. Among these markers we can cite PGE2, IL-6, IL-10, TGF-b, IDO, iNOS, HGF, KGF, TSG-6, CCL2, CMH-I, CMH-II, HLA-E. Thus, the immunomodulatory potential can for example be determined by a study of the expression of prostaglandin (PGE2) secreted by the cells in basal condition and after stimulation in an inflammatory context. For this, the cells are cultured in proliferation medium (medium with fetal calf serum) or in medium supplemented with gamma interferon, then the secreted PGE2 is measured by Elisa test (Figure 6).

The immunomodulatory potential of CSNs can also be characterized by the antiproliferative effect of CSNs on peripheral blood mononuclear cells (PBMC) treated with a mitogenic agent such as phytohemagglutinin, concanavalin A and / or lipopolysaccharides. Immunomodulation of CSNs can also be assessed by the ability of CSNs to inhibit the proliferation, secretion of pro-inflammatory cytokines and / or differentiation of T, NK, B Lymphocytes, monocytes and / or macrophages.

Thus, the immunomodulatory potential of CSNs can also be determined by the ability of CSNs to inhibit the proliferation of lymphocytes in vitro. For this, blood mononuclear cells (PBMCs) previously incubated with a fluorescent dye, are co-cultured with CSNs in CSN / PBMC ratio 1: 10 in the presence of a mitogenic agent such as concanavalin A. After 4 days of culture at 37 ° C, non-adherent cells are recovered and labeled with an anti-CD3 antibody (specific for T lymphocytes) coupled to a fluorochrome, then analyzed by flow cytometry to evaluate the signal of the fluorescent dye within the CD3 positive population. The comparison of the labeling with that carried out in a control corresponding to the PBMCs cultured in the presence of a mitogenic agent but without CSN (PBMC-ctrl), makes it possible to define an index of proliferation of the lymphocyte population. With a ratio (CSN + PBMC 1: 10) / (PBMC-ctrl) less than or equal to 0.5, CSNs are considered to exert significant antiproliferative activity (Figure 7 and Figure 8D).

The inventors have in fact demonstrated that the CSNs of phenotype CMH-I ^L / CD90 ^H according to the present disclosure has a good capacity for immunomodulation.

In a specific embodiment, the population of CSN according to the present disclosure can also be characterized in that at least 80% of the cells of said population of CSN do not have osteogenic differentiation potential. We also speak of a low potential for osteogenic differentiation or a low potential for osteogenesis. This absence of osteogenic differentiation potential or this low osteogenic differentiation potential can be evaluated by the protein and / or transcriptional study of specific bone markers (ALPL, RUNX2 ...) or by analysis of calcium deposits after induction of osteogenesis within 7 to 15 days. This osteogenic induction can be carried out by culturing CSNs in a monolayer in the presence of an osteogenic differentiation medium containing a corticosteroid, such as dexamethasone (0.1-1mM), a reducing agent such as ascorbic acid 2-phosphate (between 0 and 200pg / ml) and b-glycerophosphate (0-50mM). BMP-2 can for example replace dexamethasone.

At the end of the differentiation process, the presence of calcium deposits in the culture dish is highlighted by staining with a solution of Alizarin red 1% (weight / volume) under a microscope. The absence of deposits reveals the absence of osteogenic differentiation potential while the presence of calcium deposits colored by an intense red shows the presence of osteogenic differentiation.

Alternatively or in addition, a protein and / or transcriptional study of specific bone markers (ALPL, RUNX2) is carried out.

It should be noted that the CMH-I ^L / CD90 ^H CSNs have limited osteogenic differentiation potential compared to the CMH-I ^H / CD90 ^L CSMs with regard to the quantified markers (ALPL, RUNX2).

The inventors have been able to demonstrate that the MHC-I ^L / CD90 ^H phenotype of CSN according to the present disclosure is related to a low capacity for osteogenic differentiation of these cells.

This feature thus allows CSNs to be used in cell therapy. In particular, this is cell therapy for the treatment of arthropathy, as these limit the generation of calcified ectopic tissue within the joint. This property is particularly important for applications such as osteoarthritis, with or without tissue damage.

Pharmaceutical composition and solution for injection

Another aspect of the invention relates to a pharmaceutical composition comprising a population of CSNs as described above. Thus, the present invention also relates to a pharmaceutical composition or a ready-to-use solution for injection comprising a population of neonatal stromal cells CMH-I ^L , and optionally CD90 ^H as defined above, and a pharmaceutically acceptable vehicle.

Typically, the pharmaceutical composition or the solution for injection comprises a CSN population of 1.10 ⁶ to 1.10 ⁸ cells in a volume of 0.1 ml to 15 ml, ie a concentration of 5.10 ⁴ to 1.10 ⁹ cells / ml. In particular, it comprises from 1.10 ⁶ to 5.10 ⁷ cells in a volume of 0.1 ml to 15 ml, ie a concentration of 7.10 ⁴ to 5.10 ⁷ cells / ml. In particular, it comprises from 2, 5.10 ⁶ to 1.10 ⁷ cells for a volume of 0.1 ml to 15 ml of composition, ie a concentration of 1.5 × 10 ⁵ and 1.10 ⁸ cells / ml. In particular, it comprises from 1.10 ⁶ to 1.10 ⁷ cells in 0.5 to 2 ml, that is to say a concentration of 5.10 ⁵ to 2.10 ⁷ cells / ml.

Typically, said composition or solution for injection comprises between 1.10 ⁶ and 1.10 ⁸ cells, in particular 1.10 ⁷ cells.

By "ready to use" is meant that the pharmaceutical composition or solution for injection comprising CSNs is ready to be injected into the individual. Recultivation of the cells before use in the individual is not necessary and the cells do not need to be washed or resuspended in a physiological medium, even when they are formulated with a cryoprotector as described herein -Dessous. The expression “ready to use” means that only a thawing step is necessary when the composition or solution for injection is in frozen form, before an injection in the subject.

This pharmaceutical composition or injectable solution can be frozen, so it can be mobilized at any time. The latter has the advantage of making the treatment available as quickly as possible while limiting the human intervention necessary for its effectiveness and limiting the risk of contamination inherent in each manipulation by an operator. Thus, it is possible to separate the process for obtaining the pharmaceutical composition from its final clinical use.

Typically, the pharmaceutically acceptable vehicle and / or the packaging makes it possible to maintain the biological properties of the CSNs for a sufficient time. The vehicle can be all types of liquids, gels, solid polymers capable of containing these CSNs without damaging their desired properties, in particular an aqueous saline solution, serum, culture medium.

The packaging can be all types of receptacles, containers, medical devices capable of aseptically isolating pharmaceutical preparations from the external environment and / or from the transport environment and / or from the manipulator. In particular, the packaging makes it possible to maintain the integrity and the formulation of the pharmaceutical composition and / or to facilitate the distribution / transport of the pharmaceutical composition.

In particular, the pharmaceutically acceptable vehicle can be any solution allowing the freezing and / or thawing of cells while limiting the biological influence of these processes on cells such as the induction of cell death, differentiation, the induction of cell senescence, osmotic shock, induction of membrane porosity, modification of the membrane composition and / or phenotypic changes.

In a particular embodiment, the pharmaceutically acceptable vehicle is a solution corresponding to D-PBS (Dulbecco's phosphate-buffered saline), DMEM (Dulbecco's Modified Eagle Medium), MEM (Minimum Essential Media), a solution comprising serum of fetal calf (SVF), a solution comprising animal serum, and / or any other isotonic solution. In a particular embodiment, said solution comprises between 0 and 20% of FCS, more particularly between 5 and 20%, even more particularly 10%. In a particular embodiment, said solution is free of product of animal origin.

In a particular embodiment, said pharmaceutically acceptable vehicle is a solution comprising a cryoprotective. Insofar as the entire formulation of the solution is compatible with an injection in vivo, the solution comprising a cryoprotective agent can be used as an injectable solution for the treatments concerned by the pharmaceutical composition described in said invention.

The term “cryoprotective” means any compound making it possible to perform the cryopreservation function. According to a more particular embodiment, said cryoprotector is chosen from glycerol, dimethylsulfoxide (DMSO), propylene glycol, proteoglycans, trehalose, "Bovine serum albumin" (BSA), gelatin, polyethylene glycol (PEG) ), polyacrylic acid, poly-L-lysine, ethylene glycol or a combination of several of these cryoprotectors. In one embodiment in particular, said solution comprises between 0.5 to 30%, in particular from 0.5 to 20%, in particular from 2 to 10%, more particularly 5% of cryoprotective.

Commercial solutions comprising a cryoprotector usable in the pharmaceutical composition are synthetic pre-formulated cryoprotection solutions of the StemAlpha, CryoStor® CS2, CS5 or CS 10 type.

In a particular embodiment, said solution comprises from 0.5 to 30% of glycerol, from 0.5 to 30% of DMSO, from 0.5 to 30% propylene glycol or from 0.5 to 20% of poly- L-lysine. In particular, said solution comprising a cryoprotective is a solution comprising from 2 to 10% of DMSO, more particularly 5% of DMSO. In particular, said solution comprising a cryoprotective is a solution comprising 2 to 20% of glycerol.

In a particular embodiment, one or more adjuvants can be added such as ammonium chloride, Ringer's lactate or BSA.

In a particular embodiment, said solution comprising a cryoprotector is a DMEM solution comprising SVF, in particular from 5 to 20% of SVF and more particularly 10%.

In a particular embodiment, said solution comprising a cryoprotector is free of product of animal origin and comprises from 1 to 90% of DMSO, more particularly from 0.5 to 30%, more particularly from 2 to 10%, in particular 5%.

In another particular embodiment, said solution comprising a cryoprotector is a DMEM solution comprising SVF, in particular from 5 to 20% of SVF and more particularly 10%, and from 2 to 20% of glycerol.

In a particular embodiment, the pharmaceutical composition or solution for injection is characterized in that it is in frozen form.

The pharmaceutical composition or solution for injection can be frozen with the use of a suitable cryoprotector, capable of guaranteeing the integrity and the formulation of the pharmaceutical composition. Fa frozen pharmaceutical composition can then be stored at negative temperature between -70 ° C and -96 ° C, and more particularly at temperatures below -70 ° C for long-term storage (more than 12 months). To be used, it undergoes thawing.

The inventors unexpectedly demonstrated that the biological properties of the cryopreserved pharmaceutical composition for up to 12 months at -80 ° C. were maintained (Example E) (Figure 8A, 8B, 8C and 8D). In addition, the therapeutic efficacy of pharmaceutical composition of CSN of phenotype CMHl ^L / CD90 ^H cryopreserved has been demonstrated for the treatment of canine osteoarthritis, with an improvement in the mobility of the animal, evaluated by means of a validated questionnaire (LOAD: Liverpool Osteoarthritis in Dogs) (Figure 9), and / or by clinical evaluation by the veterinarian (Figure 10). In a particular embodiment, the inventors have also demonstrated the therapeutic effectiveness of the pharmaceutical composition of CSN of phenotype CMHl ^L / CD90 ^H cryopreserved for the treatment of thrombocytopenia (FIG. 11). The inventors have also demonstrated the therapeutic efficacy of the cryopreserved pharmaceutical composition of CSN of phenotype CMHl ^L / CD90 ^H for the treatment of chronic inflammatory diseases (IBD) (Example H).

A “ready-to-use” composition or solution for injection in frozen form according to the invention can thus be mobilized at any time. The latter has the advantage of making the treatment available as quickly as possible while limiting the human intervention necessary for its effectiveness and limiting the risk of contamination inherent in each manipulation by an operator. Thus, it is possible to separate the process for obtaining the pharmaceutical composition from its final therapeutic use.

In one embodiment, the present invention relates to a ready-to-use injectable solution comprising a population of CSN CMH-I ^L , and optionally CD90 ^H , as described above and a cryoprotective agent as defined above.

Typically said population of CSN and said cryoprotector are formulated in a pharmaceutically acceptable vehicle as defined above.

In a particular embodiment, said ready-to-use injectable solution comprises a unit dose of CSN CMH-I ^L , and optionally CD90 ^H , of 1.10 ⁶ to 1.10 ⁸ cells, in particular 1.10 ⁷ cells, and a solution comprising a cryoprotector as defined above.

In a particular embodiment, said injectable solution comprises a unit dose of 1.10 ⁶ to 1.10 ⁸ cells in a volume of 0.1 to 15 ml, more particularly of 1.10 ⁶ to 1.10 ⁷ cells, typically 1.10 ⁷ cells, in a volume of 0.5 to 2 ml.

In a particular embodiment, at least 80% in number, in particular at least 85%, more particularly at least 90% of the CSNs are of phenotype CMH-I ^L / CD90 ^h . In a more particular mode, the CSNs are placental CSNs, more particularly of canine origin. In a particular embodiment, the cryoprotective in said injectable solution is DMSO. In a particular mode, the solution for injection is free of product of animal origin and comprises from 1 to 90% of DMSO, more particularly from 0.5 to 30%, more particularly from 2 to 10%, in particular 5%.

In a particular embodiment, the CSNs can undergo exogenous stimulation / modification before injection by means of physical, biological and / or chemical effectors. By exogenous stimulation (also called "priming") we mean all stimulation / modifications of cells and / or their microenvironment triggering in them a phenotypic change favoring their biological properties within the framework of specific therapies. For example, treatment with cytokines in concentrations between 1 and 500 ng / ml of IFN-g, IL-1b, 11-6 and / or TNF-a makes it possible to significantly increase the expression of molecules having immunomodulatory activity. In another example, mechanical stimulation and / or the induction of a chondrogenic predifferentiation can make it possible to favor the tissue reconstruction properties in vitro.

Neonatal stromal cell

Another aspect of the disclosure relates to a neonatal stromal cell of phenotype CMH-I ^l , in particular of phenotype CMH-I ^L / CD90 ^h .

This cell can also be characterized structurally by the fact that it does not express one or more of the following surface markers: CD1 lb, CD14, CD31, CD34, CD45, or the HLA-DR also called CMH-II.

Furthermore, functionally, it can be characterized in that it has:

an adhesion capacity on a plastic support; and or

a consecutive cell doubling capacity greater than 20 cumulative doublings, and / or

a potential for chondrogenic differentiation, and / or

an immunomodulatory potential; and or

little or no osteogenic differentiation potential.

In a particular embodiment, the present disclosure relates to a neonatal stromal cell for its therapeutic use in dogs, cats, horses, or humans, for example for the treatment: at. tissue or osteo-articular damage, with or without an inflammatory component;

b. degenerative diseases, including osteoarthritis, tendinopathies, tissue fibrosis, Alzheimer's, Parkinson's;

vs. autoimmune, inflammatory and / or infectious diseases, especially atopic dermatitis, gingivostomatitis, thrombocytopenia, epidermolysis bullosa, sepsis, inflammatory bowel disease (IBD);

d. transplant rejection, or

e. tumor diseases.

Such a CSN cell can advantageously be isolated from the population of CSN cells according to the present disclosure as described above, according to the cloning methods known to those skilled in the art.

Therapeutic use

Another aspect of the present disclosure relates to a population of CSN, a pharmaceutical composition or an injectable solution as defined above for its therapeutic use.

Typically, this is for use in cell therapy. By "cell therapy" is meant a therapeutic treatment comprising the administration of cells capable of inducing a beneficial therapeutic effect in the individual. As part of a regenerative medicine approach, this cell therapy is likely to directly (cell differentiation) or indirectly (secretion of biological factors, activation or inhibition of environmental cells) promote the in vivo regeneration of one or more biological tissue in an individual awaiting such treatment.

In particular, it is a use in the same individual, or in an individual of the same species, or in an individual of a different species compared to the species from which said CSN originates.

When the recipient subject is identical to the individual from whom the CSNs come, we speak of an autologous therapeutic use. When the recipient subject is an individual of the same species as the species from which the CSN originates, this is called heterologous or allogenic therapeutic use.

When the recipient subject is an individual of a different species compared to the species from which the CSN originates, this is called xenogenic therapeutic use.

In a particular embodiment, the present invention relates to a population of CSN as defined above for its xenogenic therapeutic use.

In a particular embodiment, the present disclosure relates to said population of CSN, a pharmaceutical composition or an injectable solution as defined above for its therapeutic use in a mammal. In particular, said mammal is a dog, a cat, a horse, or a human.

For example, therapeutic use can be the treatment:

at. tissue or osteo-articular damage, with or without an inflammatory component;

d. transplant rejection, or

e. tumor diseases.

Tissue damage is understood to mean damage, loss of normal function and / or deterioration induced by excessive solicitation of the tissue, normal solicitation of pathological tissue or all tissues requiring tissue reconstruction / healing: myocardial infarction, renal damage, liver damage, burn, skin lesions, fractures, respiratory damage, osteo-articular damage such as dissecting osteochondritis. By degenerative disease, we mean all types of diseases where the homeostatic balance is deregulated in favor of an exacerbated tissue catabolism or an excessive induction of tissue anabolism, such as for example: osteoarthritis, tendinopathies, tissue fibrosis, Alzheimer, Parkinson. CSNs can also be used in the treatment of diseases or physiological disturbances linked to an unwanted immune response, i.e. all types of diseases where the immune system interferes with the normal / physiological functioning of the tissue or with a treatment aimed at treating an immune disease and / or reducing the normal / physiological functioning of a tissue, such as atopic dermatitis, gingivostomatitis.

They can be used in the treatment of autoimmune and inflammatory diseases such as the graft versus host reaction (graft versus host disease or GvHD), tissue or organ transplant, autoimmune diseases such as multiple sclerosis, inflammation tissue, allergy, asthma, allergic bronchitis, chronic bronchitis such as chronic obstructive pulmonary disease (COPD), chronic inflammatory bowel disease, kidney failure, thrombocytopenia, lupus erythematosus, rheumatoid arthritis, epidermolysis bullosa.

"Inflammatory bowel disease (IBD)" means chronic inflammations of the mucous membranes of the small intestine, the colon and the idiopathic anoperineal region, such as duodenal enteritis in dogs and cats , and more particularly inflammations such as Crohn's disease and ulcerative colitis in humans. These conditions are characterized by gastrointestinal and chronic disorders associated with inflammatory mucosal infiltration. They are most often diagnosed with vomiting and chronic diarrhea in animals and humans. IBD is distinct from enteropathies responding to dietary change and diarrhea responding to antibiotics. By definition, they respond to immunosuppressive agents and not to a specific diet or antibiotics. Clinical signs such as vomiting, diarrhea, weight loss, loss of appetite are due to cellular mucosal infiltrates, mediators of inflammation, enterocyte dysfunction associated with inflammation and bowel motility disorder.

The CSNs according to the invention can also be used in the treatment of diseases in which chronic inflammation linked or not to an immune disturbance causes tissue degeneration or a dysfunction of the function of an organ or a tissue such as arthritis, tendonitis.

On the other hand, said population of CSN, pharmaceutical composition or an injectable solution can be used in the treatment of infectious diseases associated or not with the preceding components. By infectious diseases we mean diseases involving contamination by pathogens such as microorganisms such as protozoa, bacteria, and / or viruses. These infectious diseases can be localized or systemic in nature. The use of NSCs can in particular be prescribed in the specific context of antibiotic resistance of the pathogens involved and in the context of a generalized inflammatory response associated with a serious infection, such as sepsis.

The therapeutic framework can extend to diseases with several pathological aspects such as osteoarthritis with a degenerative side and an inflammatory side. Said population of CSN, pharmaceutical composition or an injectable solution can also be used in the treatment of diseases of a tumor nature with or without a metastatic component.

The therapeutic framework can also extend to the use of NSCs in a combined way with other types of therapies such as for example: laser; shock waves; platelet rich plasma (PRP); hyaluronic acid ; non steroidal anti inflammatory.

In a particular embodiment, the present disclosure relates to a population of CSNs for use in tissue engineering. By "tissue engineering" is meant all biotechnology techniques using cells and biomaterials (of biological or synthetic origin) to generate tissue substitutes in vitro / ex vivo for implantation in vivo or to be used as a model tissue in the laboratory. (Ex: reconstruction of the skin, bone or cartilage).

In a particular embodiment, the present disclosure relates to a population of CSN, a pharmaceutical composition or an injectable solution as defined above for its use in the treatment of indications of the musculoskeletal system such as osteoarthritis in mammals, more particularly in dogs, cats or horses, preferably in dogs.

In another particular embodiment, the present disclosure relates to said population of CSN, pharmaceutical composition or injectable solution as defined above for its use in the treatment of thrombocytopenia in mammals, more particularly in dogs, cats or horses , preferably in dogs.

In a particular embodiment, the present disclosure relates to said population of CSN, pharmaceutical composition or solution for injection as defined above for its use in the treatment of inflammatory diseases chronic bowel disease, such as duodenal enteritis in mammals, more particularly in dogs, cats or horses, preferably in dogs.

In a particular embodiment, the present disclosure relates to said population of CSN, pharmaceutical composition or injectable solution as defined above for its use in the treatment of inflammatory bowel disease, such as Crohn's disease in humans. .

Processing method

According to another aspect, the present invention relates to a treatment method:

d. transplant rejection, or

e. tumor diseases,

comprising the administration of said population of CSN or of a pharmaceutical composition or an injectable solution as defined above in the subject to be treated.

In particular, the present invention relates to a method of treatment of indications of the musculoskeletal system such as osteoarthritis in mammals, more particularly in dogs, cats or horses, preferably in dogs, comprising the administration of said population of CSN or of a pharmaceutical composition or an injectable solution as defined above in the subject to be treated.

In particular, the present invention relates to a method of treatment for the treatment of thrombocytopenia in mammals, more particularly in dogs, cats or horses, preferably in dogs, comprising the administration of said population of CSN or a pharmaceutical composition or an injectable solution as defined above in the subject to be treated. In particular, the present invention relates to a method of treatment for the treatment of chronic inflammatory diseases of the intestine in mammals, more particularly in dogs, cats or horses, preferably in dogs, comprising the administration of said population of CSN or of a pharmaceutical composition or an injectable solution as defined previously in the subject to be treated.

Administration mode

The population of CSN, a pharmaceutical composition or an injectable solution as defined above, can be administered locally or by intravenous (IV) route or more generally by parenteral route.

Typically, a local administration can be an administration by intra-articular injection, for example in the case of the treatment of indications of the musculoskeletal system such as osteoarthritis, at the level of each joint to be treated.

By IV administration is meant administration of the CSNs directly into the subject's venous system using a catheter or needle or, for example, via an infusion bag or, for example, through the infusion set. Typically, intravenous administration is performed in the treatment of thrombocytopenia, osteoarthritis, or the treatment of inflammatory bowel disease such as duodenal enteritis or Crohn's disease.

In a particular embodiment, 1.10 ⁶ to 1.10 ⁸ CSN, in particular 2.5.10 ⁶ to 1.10 ⁷ CSN, more particularly 1.10 ⁷ CSN, are administered intra-articularly, at the level of each joint to be treated, for the treatment indications of the musculoskeletal system such as osteoarthritis.

In a particular embodiment, 1.10 ⁶ to 10.10 ⁸ CSN, in particular 1.10 ⁷ CSN, are administered intravenously for the treatment of inflammatory diseases with a dysimmunity component, such as thrombocytopenia.

In a particular embodiment, 1.10 ⁶ to 5.10 ⁶ CSN / kg are administered intravenously for the treatment of inflammatory diseases with a dysimmune component, such as thrombocytopenia.

In a particular embodiment, 1.10 ⁶ to 10.10 ⁸ CSN, in particular 1.10 ⁷ CSN, are administered intravenously for the treatment of inflammatory bowel disease. The mode of administration is obviously adapted according to the subject and the pathology to be treated. The exact number of cells to be administered depends on various factors, including the age, weight, sex of the subject to be treated, the condition and the extent or severity of the condition to be treated.

Obtaining process

The present disclosure also relates to an in vitro process for obtaining a pharmaceutical composition of neonatal stromal cells derived from neonatal tissues (FIG. 16 and FIG. 17), said pharmaceutical composition comprising as active principle a population of neonatal stromal cells comprising phenotype of CNS MHC-I ^l, and optionally phenotype CD90 ^H, said method comprising:

at. the supply of one or more neonatal biological samples comprising CSNs, the biological sample or samples having been previously obtained from one or more individuals,

b. isolation of the CSN population present in the biological sample (s),

vs. optionally, at least one step of ex vivo amplification of the CSNs obtained in step b.,

d. optionally, cryopreservation of the CSN population obtained in step b. or c.,

e. optionally, stimulation by physical, biological and / or chemical effector of the CSN population obtained in step b., c. or d.,

f. the characterization of the presence of CSN of phenotype CMH-I ^L , and optionally of a phenotype CD90 ^H , among at least 80% of the population of CSN, after isolation in step b. and / or after the ex vivo amplification step in step c. and / or after cryopreservation of the CSNs in step d.,

g. suspending the population of CSN comprising at least 80% of CSN of phenotype CMH-I ^L , and optionally of phenotype CD90 ^H , in a pharmaceutically acceptable suspension medium.

In one embodiment, the neonatal biological sample (s) provided in step a., Come from a neonatal tissue sample, in particular from one or more placentas and / or from one or more umbilical cords, or from one or more membranes amniotics, or a sample of neonatal fluid such as for example the amniotic fluid of one or more amniotic sacs and in particular the blood of one or more umbilical cords. In a particular embodiment, the neonatal biological sample is a placenta.

This sample can be taken as previously explained in the request.

In particular, the neonatal biological sample (s) provided in step a) of the process come from mammals, and more particularly from dogs, cats, horses or humans. In a particular embodiment, the neonatal biological sample is a sample from the dog, also called a canine sample.

In a particular embodiment, the neonatal biological sample (s) provided come from the same individual, from one or more individuals or from a mammalian model different from that for which the pharmaceutical composition is intended.

By "isolation" is meant the operation of extracting by an enzymatic and / or mechanical process, the cells contained in a tissue and its extracellular matrix.

The isolation of CSNs is carried out from a neonatal tissue, for example, by dissection and enzymatic digestion of the tissue, then by centrifugation and recovery of the cell pellet containing CSNs. Alternatively, it is made from a sample of umbilical cord blood. The blood cells can then be separated on a density gradient, in particular using Licoll. The cell ring formed at the interphase between the diluted plasma and the Licoll is recovered, and the cells are washed and centrifuged, then the cell pellet containing CSN is recovered. The cells are generally counted and seeded at a density between 10 ⁵ and 5.10 ⁵ cells / cm2. The number of total cells recovered after centrifugation can be for example between 0.1.10 and ⁶ 500.10 ⁶ cells, specifically in dogs between ⁶ and 10.10 ⁶ 0.1.10, specifically in horses and 100.10 ⁶ 500.10 ^6.

Following the isolation of cells containing a fraction of CSN, a step of amplification of CSN can be carried out by adhesion to the plastic.

In order to obtain larger amounts of CSN for the purpose of making different pharmaceutical preparations, isolated CSNs can undergo an amplification step in the laboratory.

By “amplification step” is meant any step allowing proliferation of CSNs on plastic or polymer support. This phase must be capable of promoting the presence of CSNs to the detriment of other cell types which do not meet the characteristics of CSNs. It must also ensure optimal proliferation of cells while limiting the phenomena of dedifferentiation, differentiation and / or senescence. This step involves conditions in a controlled atmosphere such that a person skilled in the art is able to establish, for example with 90% humidity and comprising 5% C0 ₂ . The amplification temperature must be constant and between 35-40 ° C, more precisely between 37-39 ° C. Among the culture media, in a non-exhaustive manner, it is possible to cite the media of the Alpha-MEM, DMEM, RPMI, IMDM, Opti-MEM, EGM, EGM-2 type, synthetic media adapted to the culture of MSCs lacking endotoxin and / or serum, synthetic media adapted to good manufacturing practices, whether or not supplemented with fetal calf serum (SVF) from 0.1% to 20%, platelet lysate, insulin -transferin-selenium , defined commercial supplements and / or other growth factors and / or molecules promoting the proliferation of CSNs while limiting their senescence such as FGF, EGF, VEGF, dexamethasone and / or A2P.

This amplification phase can be carried out on different supports once the CSN population is obtained following the isolation step. These different supports can be 2D or 3D in nature.

By amplification on 2D support is meant all cell culture methods allowing amplification of CSNs on monolayer support and properly developed by those skilled in the art. In particular embodiments, the cells can be cultured in plastic culture dishes treated or not to promote cell adhesion, of flaccid type, with one or more stages and / or of multilayer type with or without continuous perfusion, with or without optimizing the air flow.

By amplification on a 3D support is meant all techniques known to those skilled in the art using biomaterials, microcarriers and / or polymers capable of ensuring amplification of the CSNs in a bioreactor and correctly developed by those skilled in the art. art. In particular embodiments, the CSNs can be amplified in shaking, axial and / or pendulum bioreactors, in wave shaking, in rotating bed shaking, in static and / or infused bioreactors. The biomaterials and / or microcarriers can be of several types and according to particular embodiments can be of sizes between 100-500 mhi in diameter, have a porosity of different nature, have a treated surface, whether or not negatively or positively charged, include growth factors or recombinant proteins of the integrin and / or extracellular matrix type or any other biological / chemical molecules promoting cell adhesion and / or cell proliferation.

Since the CSNs are adherent cells, in order to ensure the amplification step, cell passage of the CSNs may be necessary and carried out by a method correctly developed by a person skilled in the art. A cell passage (P) corresponds to the detachment of cells from their support when they arrive at confluence (cell mat), to put them back in culture on a new support. Typically, the detachment of cells can be carried out under the effect of mechanical action, enzymes and / or inhibitors such as, in a non-exhaustive manner, recombinant or animal trypsin, EDTA and / or accutase. It is also possible to carry out these cellular passages by the use of dissolvable biomaterials / microcarriers according to a process perfected by those skilled in the art.

In a particular embodiment, for the amplification phase, the cells are for example treated with trypsin-EDTA, then taken up in an amplification medium and centrifuged. After taking up in amplification medium, they are re-cultured at the level of 1500 to 5000 cells / cm ² or 3D equivalent in amplification medium with or without monitored monitoring of the microenvironment and / or culture atmosphere.

In a particular embodiment, the amplification step can comprise several cellular passages.

During this amplification stage, the CSNs multiply by doubling cell. Thus, the amplification step can also be defined in terms of cell doubling.

In a particular embodiment, said method comprises an amplification step in which the population of CSN according to the invention undergoes 2 to 25 cell doublings, more particularly 5 to 15 cell doublings.

After isolation from neonatal tissue, cells can be cryopreserved in cellular seed units. For this, the cells are centrifuged and then taken up in a freezing medium. The freezing medium can either be a culture medium such as for example DMEM enriched with 5-50% of SVF (vol: vol) and 5-10% (vol: vol) of DMSO or a commercial cryopreservation medium, containing or not a fraction of DMSO. The cells are frozen, for example, under a controlled temperature drop (- l ° C / min up to a temperature of -80 ° C), using for example a CoolCell® Cell Freezing Containers (BioCision) or a programmable freezer of the Digitcool type (Cryobiosystem) Fes CSN thus frozen can be stored in particular at temperatures below -70 ° C for long-term storage (more than 12 months).

The stage of characterization of the presence of a CMH-I ^L phenotype, and optionally of CD90 ^H phenotype, is carried out according to the previously described methods of single labeling or double labeling. This stage is carried out on a sample of the population of CSN resulting from stage b., C. and / or d., to characterize said population. The purpose of this step is to select a population of CSN comprising at least 80% of its cell population of phenotype CMH-I ^L , and optionally of phenotype CD90 ^H. The CSN populations meeting this criterion are selected and can be suspended in a pharmaceutically acceptable suspension medium, such as for example a buffered saline solution, a sterile injectable solution containing 10-100 USP of heparin sodium, a cryopreservation medium. commercial used as an excipient.

In a particular embodiment, the characterization step consists in the characterization of CSN CMH-I ^L and CD90 ^H , and is carried out by double labeling of the markers CMH-I and CD90 in flow cytometry, as described above. The characterization step of the presence of a MHC-I ^L / CD90 ^H phenotype corresponds to an evaluation of rMFI MHC ^L less than 20, more particularly less than 15, more particularly less than 10 and according to an evaluation of rMFI CD90 greater to 15 and more particularly greater than 20, among at least 80% of the CSNs of the CSN population.

In a particular embodiment, said method comprises an amplification step c., Said amplification step comprising 1 to 5 cell passages, more particularly comprising 2 or 3 cell passages. In this embodiment, the characterization of the population of CSNs isolated in step b. is therefore carried out after an amplification step comprising 1 to 5 cell passages (step c.), more particularly comprising 2 or 3 cell passages.

In one embodiment, said method comprises an amplification step c., Said amplification step said amplification step corresponds to a doubling of the population of CSNs isolated in step b. from 2 to 25 cell doublings, in particular 5 to 15 cell doublings. In this embodiment, the characterization of the population of CSNs isolated in step b. is therefore carried out after an amplification step corresponding to a doubling of the population of CSNs isolated in step b. from 2 to 25 cell doublings, in particular 5 to 15 cell doublings.

In one embodiment, said amplification step can be characterized in terms of passages and in terms of cell doublings as defined above.

The inventors have found that between passage 1 (Pl) and passage 5 (P5), in particular between passage 2 (P2) and passage 3 (P3), an important phenotypic change takes place. In fact, they discovered that, surprisingly, the phenotypic profiles of the different CSN populations are similar between P0-P1 and are mainly of the MHC-1 ^H and CD90 intermediate type. On the other hand, between Pl and P5, more particularly between P2 and P3, an individualization of the CSN subpopulations of different phenotype is observable. By “individualization”, it is meant that two CMH-I ^L / CD90 ^H and CMH-I ^H / CD90 ^L subpopulations are observable within the same CSN population, during cell amplification. Indeed, during the in vitro amplification between Pl and P5 and more particularly between P2 and P3, the CSN populations go through an individualization phase where the two sub-populations CMH-I ^H / CD90 ^L and CMH- I ^L / CD90 ^H are easily identifiable in the case of a heterogeneous population.

In an alternative embodiment, it is possible to combine the notion of passage with the number of doublings: the individualization phase can then be considered between 2 and 25 doublings, in particular 5 to 15 doublings.

Typically, the characterization of the expression of the markers is carried out by the double marking method described above.

If this individualization is observable, an analysis matrix specific to the isolated CSN population of phenotype CMH-I ^L / CD90 ^h , can be determined in order to be used for the analysis of the sub-populations that it contains and / or therapeutic units and banks produced from this same population of CSN (Figure 16 and Figure 18). By analysis matrix is meant an analysis window determined for the analysis in flow cytometry. The purpose of this matrix is to define the stage of individualization and / or the phenotype of a population of given CSNs, during the amplification stages and / or after thawing and / or during the manufacturing process of the pharmaceutical preparation. .

The exploitation of this matrix as a characterization tool involves an analysis at least on the first pass, in particular at least on the second pass to correctly assess the proportion of the different CSN subpopulations during the manufacturing process.

In a particular embodiment, this phase of individualization and the establishment of an analysis matrix can be used in order to adjust the determination of thresholds previously described, for the characterization of the expression of the markers. This phenomenon can be exploited to determine the rMFI of MHC-I and CD90 for each of the subpopulations within the mother population (Figure 3). The analysis, also, of a sufficiently large number of heterogeneous CSN populations can make it possible to establish thresholds with more precision for the determination of the phenotype CMH-I ^L / CD90 ^H during the manufacturing process of the invention .

In a particular embodiment, the process for obtaining the pharmaceutical composition of CSN comprises a step of enriching the population of CSN in CSN of phenotype CMH-I ^L , and optionally of phenotype CD90 ^H , which can correspond to a depletion step of CSN cells of phenotype CMH-I ^L , and optionally of phenotype CD90 ^H. This enrichment step by cell depletion can be carried out by cell sorting methods of column chromatography, by the use of magnetic beads coupled to antibodies of interest, or by flow cytometry coupled to a cell sorter.

In a particular embodiment, the process for obtaining the pharmaceutical composition of CSN can also comprise a step of inhibiting MHC-I, for example by in vitro modification of the phenotype of the cells (extinction of a gene) or by masking receptors for example using an antibody.

At the end of the process, the preparation of CSN can then be amplified in vitro, cryopreserved or administered to an individual suffering from one of the pathologies mentioned above. During the process, it is possible to carry out a cryopreservation step, after the suspension of the CSNs in a pharmaceutically acceptable suspension medium comprising a cryoprotective. By “cryopreservation” is meant the step of storing frozen cells for a period ranging from 1 day to 5 years and more. The cell bank is conditioned so as to guarantee the integrity and the biological properties of the cell populations.

The cryopreservation step is preceded by a freezing step corresponding to the descent in temperature of the cell suspension to its storage temperature.

In a particular embodiment, this freezing step corresponds to a gradual descent of the temperature of the cell suspension (-1 ° C / minutes) to reach the storage temperature ranging from -70 ° C to -196 ° C. In an alternative embodiment, the cells can be frozen at a freezing rate between - 0.3 ° C / minutes and -99 ° C / minutes. The same freezing protocol can include one or more different freezing speeds as in the case of a gradual increase in freezing speed. In an alternative embodiment, the cells are directly frozen without temperature control in a storage enclosure whose temperature is between -70 ° C and 196 ° C. Final storage can be in the liquid phase (of the liquid nitrogen type) or the gas phase (of the enclosure type-140 ° C., -80 ° C. or nitrogen storage in the gas phase).

In order to make the cryopreserved cells available for therapeutic administration to the subject, a thawing step is carried out following the freezing step, in the case where the CSNs used for the administration are in the form of bank units. This thawing is carried out so as to pass from the stage of frozen cells to the stage of thawed cells during an embodiment limiting cell death by drying, mechanical damage to the plasma membrane, osmotic shock. In a particular embodiment, the CSN units are heated by manual friction for less than 10 min. In another particular embodiment, the CSN units are placed in a liquid or dry water bath, set at a temperature between 30 and 40 ° C for less than 10 min, in particular at 37 ° C for 3 to 5 min. In another particular embodiment, the CSN units are placed in an automatic thawing apparatus. In another particular embodiment, the CSN units are thawed at room temperature for less than 10 min if the cryoprotector used allows it. CSN population obtained by the process

The present invention also relates to a CSN population as defined above, obtained by a method comprising:

b. isolation of the CSN population present in the biological sample (s),

vs. a step of ex vivo amplification of the CSNs obtained in step b.,

In a particular embodiment, said amplification step comprises 1 to 5 cell passages, more particularly comprising 2 or 3 cell passages.

In a particular embodiment, said amplification step corresponds to a doubling of the population of CSNs isolated in step b. from 2 to 25 cell doublings, in particular 5 to 15 cell doublings.

After characterization, said CSN population can be formulated as a pharmaceutical composition or solution for injection, as described above.

FIGURES

Figure 1: Parameterization and cytometric analysis of CSN.

(A) Definition of an analysis window based on the FSC-A / SSC-A analysis parameters, then of an analysis window based on the FSC-H / FSC-A analysis parameters. (B) A 2D representation (2 parameter histogram) is used on the basis of the analysis of the fluorescence from the fluorochrome used (FL: 2 for PE) and of the auto fluorescence of the cells using a fluorescence channel with an excitation / emission spectrum not used in the analysis (FL-4). An analysis window is defined on the samples marked with the isotype, for a negativity tolerance threshold of around 0.5%. (C) Analysis of the CMH-I marker. (D) Analysis of the CD90 marker. (E) A representation by superposition of the histograms is carried out while preserving the same parameters.

Figure 2: Demonstration of differences in MHC-I expression in CSNs.

Based on the configuration and analysis presented in Figure 1, several samples of CSN are analyzed. Analysis of the expression of the MHC-I indicates variable expressions of this marker between the different samples. The proposed analytical method consists in normalizing the mean fluorescence values (MFI; Mean Fluorescence Intensity) of the cells labeled with the antibody of interest (here MFI FL2 (CMH-I-PE)) with the MFI of the cells labeled with the isotype coupled to the same fluorochrome (here MFI FL2 _(isotype-PE) ) · A ratio is thus calculated MFI FL2 (CMH-I-PE) / MFI FL2 (isotype-PE). (A) Analysis of the CMH-I marker. The acceptability threshold for defining MHC-I negative cells was set at 2.5. (B) The parameters and the analyzes are carried out in accordance with the description of FIG. 1 with the difference that a secondary antibody coupled to a fluorophore of allophycocyanin type (APC) is used. The cells marked with an isotype (CSN-ISO) and with the MHC-I (CSN-MHC-I) are presented. In the same way, the analytical method consists in normalizing the mean fluorescence values (MFI; Mean Fluorescence Intensity) of the cells labeled with the antibody of interest (here MFI FL4 (CMH-I-APC)) with the MFI of the cells. marked with the isotype coupled to the same fluorochrome (here MFI FL4 (isotype-APC)). A ratio is thus calculated MFI FL4 (CMH-I-APC) / MFI FL4 (isotype-APC). The acceptability threshold for defining MHC-I negative cells was set at 10.

Figure 3: Comparison of the expressions of the CMH-I and CD90 markers between the two subpopulations present in heterogeneous (mixed) CSN populations.

CSNs are isolated from canine placenta and analyzed by flow cytometry between P1-P4. When a mixed population is identified, the expressions of MHC-I and CD90 are evaluated in each of the respective sub-populations MHC-I ^H / CD90 ^L (MHC-I / CD90 - HL) and MHC-I ^L / CD90 ^H ( MHC-I / CD90 - LH). The results are presented in the form of relative MFIs (rMFI). The analysis makes it possible to establish with precision the expression thresholds of CMH-I and CD90 for the population of interest CMH-I ^L / CD90 ^H (n = 13). Figure 4: Difference in proliferation potential between CSNs of type CMH-I ^L / CD90 ^H and CMH-I ^H / CD90 ^l .

CSNs are isolated from canine placenta and analyzed by flow cytometry on several passages between P2 and P7. An analysis matrix presented in FIG. 18 is determined for each population of isolated CSNs having presented the two subpopulations CMH-I ^L / CD90 ^H and CMH-I ^H / CD90 ^L during culture. The CSNs are characterized as CMH-I ^L / CD90 ^H when the median analysis of the percentages of the CMH-I ^L / CD90 ^H subpopulation (passages between P2-P7) represents at least 75% of the total population. The results of the maximum number of doublings and the average doubling time are presented in the form of a boxplot (CMH-I ^L / CD90 ^H n = 4; CMH-I ^H / CD90 ^L n = l 1). The probability p of H0 rejection is determined by the Mann-Whitney U-test.

Figure 5: Difference in chondrogenic potentials between CSNs of type CMH-I ^L / CD90 ^H and CMH-I ^H / CD90 ^l .

A CSN-1 population of CMH-I ^L / CD90 ^H phenotype and two populations of CSN-2 and CSN-3 of CMH-I ^H / CD90 ^L phenotype were studied after 7 days of chondrogenic differentiation. (A) Observation of micromasses with a photonic microscope (magnification x4). (B) Histogram representing the surface of the micromasses analyzed under ImageJ and expressed in pixels ² according to the cell population. (C) Expression of the Col2al gene in the micromasses obtained for each cell population after 7 days of chondrogenic differentiation (treated) compared to this same cell population which has not undergone chondrogenic differentiation (Ctrl): The expression levels are presented in relative expression of Col2al mRNA. (D) CSNs are isolated from canine placenta and analyzed by flow cytometry on several passages between P2 and P7. An analysis matrix presented in FIG. 18 is determined for each population of isolated CSNs having presented the two sub-populations CMH-I ^L / CD90 ^H and CMH-I ^H / CD90 ^L during culture. The CSNs are characterized as CMH-I ^L / CD90 ^H when the median analysis of the percentages of the CMH-I ^L / CD90 ^H subpopulation (passages between P2-P7) represents at least 75% of the total population. The results of the determined chondrogenic potential as described in the invention are presented (CMH-I ^L / CD90 ^H n = 3; CMH-I ^H / CD90 ^L n = 5). The probability p of rejection of HO is determined by the Mann-Whitney U-test. Figure 6: Determination of PGE2 in culture supernatants of CSN CMH-F (2 independent lines) after 3 days of culture in basal condition (CTRL) or after 3 days of treatment with 5ng / ml of gamma interferon (IFN)

Figure 7: Evaluation of the antiproliferative effect of CSN on T lymphocytes in vitro

This figure illustrates the antiproliferative effect of CSNs after 4 days of co-culture of CSNs with PBMCs (1: 10 ratio) in the presence of a mitogenic agent (mitomycin). The control (CTRL) is a culture of PBMCs under the same conditions in the presence of a mitogenic agent but without CSN. Analysis of lymphocyte proliferation is determined by evaluating the signal of a fluorescent marker (Celltrace) in the population marked with an anti-CD3 antibody (specific marker for T lymphocytes) coupled to a fluorochrome. A proliferation index (PI) is calculated for each experimental condition using the Modfit® analysis software. The IP in the presence of CSN is normalized compared to the IP in control condition, set to 1.

Figure 8: Evaluation of the biological properties of the post-thawed cryopreserved pharmaceutical composition

(A): Evaluation of the cellular viability of the CSNs before cryopreservation (CTRL culture; n = 3) and after thawing of the cryopreserved cells maintained 3, 6, 9, and 12 months at -80 ° C. (3M, 6M, 9M, 12M ; n = 3 for each condition),

(BC): The proliferative activity of CSNs from a subculture (CTRL culture; n = 6) or from cryopreserved units for 3, 6, 9, 12 months (3M, 6M, 9M, 12M; n = 3 for each condition) was evaluated by seeding 225,000 cells in a T75 flask, maintained in culture in an incubator for 7 days. The amplification medium is renewed once during the culture. The number and the doubling time are calculated according to the formula described in the example. The results do not show a significant difference in the number and doubling time between the CSNs maintained in culture and the cryopreserved CSNs, up to 12 months of storage at -80 ° C. (D): The analysis of lymphocyte proliferation in co-culture with cultured CSNs (n = 3) or cryopreserved CSNs (n = 6) does not reveal any significant difference in the antiproliferative effect of CSNs.

Figure 9: Evaluation of the mobility of dogs before and after administration of cryopreserved CSNs based on the LO AD questionnaire. Two arthritic dogs are treated with 1.10 ⁷ of CSN by joints of phenotype CMH-I ^L / CD90 ^H cryopreserved and thawed at room temperature. The clinical evolution of the subjects is carried out by analysis of the LO AD score after treatment as described in Example E. The two subjects presented showed a reduction in the LO AD score following treatment with the cells.

Figure 10

15 arthritic dogs are treated with 1.10 ⁷ of CSN by articulation of phenotype CMH-I ^L / CD90 ^H cryopreserved and thawed at room temperature. The clinical monitoring of dogs is carried out six months post-injection. 87% of the cases showed a satisfactory evolution following the treatment.

Figure 11

A dog diagnosed as having thrombocytopenia is treated with 1.10 ⁷ of CSN of phenotype CMH-I ^L / CD90 ^H cryopreserved and thawed at room temperature. The cells are injected by intravenous infusion. The results show a normalization of platelet concentrations over 3 months. The treatment also made it possible to get rid of corticosteroid treatments.

Figure 12: Representative examples of the different cytometric profiles and their associated characteristics.

CSNs are isolated from canine placenta and amplified in several passages (2 to 5). Several CSN samples are analyzed by flow cytometry to assess the expression of CD90 (FL2-PE) and MHC-I (FL4-APC). Depending on the CD90 / CMH-I phenotypic profile, CSNs can be classified into several categories according to their biological characteristics and their composition. The implementation of the analysis matrix is presented in Figure 18.

Figure 13: Heterogeneity of expression of CMH-I and CD90 among the amplified CSNs

NSCs are isolated from canine placenta, equine umbilical cord blood and equine umbilical cord matrix (n = ll, 13 and 18 respectively). The CSNs were amplified at least on one pass and analyzed by flow cytometry to evaluate their percentage of positivity and the rMFI ("relative mean of fluorescence intensity") for the MHC-I and the CD90. This figure illustrates the disparities in markings and expression for these two markers among the CSNs from the same source.

Figure 14: Representative example of positivity fluctuations as a function of different thresholds for weak marker expressions.

CSNs are isolated from canine placenta, amplified and analyzed by flow cytometry. The expression of CD90 is evaluated by simple labeling in FL2 (Fluorescence PE). The positivity thresholds are placed at around 2 (CD90pos_2SD) or 3 (CD90pos_3SD) standard deviations of the PE isotypic population, ie 4.5% or 0.3% of the PE isotypic population respectively. This figure shows the amplitude of the results as a function of the two thresholds established for the same population of CSNs weakly expressing CD90 (between 89.0 and 65.3%).

Figure 15: Disparity of proliferative potential among CSN populations from canine placentas. CSNs are isolated from canine placenta and amplified over several passages until a doubling time greater than 100 h is obtained. At each pass, the number of cell doubling and the cell doubling time is calculated according to Example C, part 1. The maximum number of doubling is then determined over the entire amplification period. The doubling time is averaged over 3 passages between P2 and P4 so as to obtain data representative of the proliferation potential of the CSNs.

Figure 16: Flow diagram of the manufacturing process for CSN CMH-I ^L / CD90 ^H for therapeutic use - manufacturing process assisted by analysis matrix.

Organizational chart allowing both to determine the CMH-I / CD90 analysis matrix, to collect the analysis data and allowing a double control conditioning the industrialization of therapeutic units and their use. The condition “True” implies that a population of CSN is qualified as CMH-I ^L / CD90 ^H according to the invention.

Figure 17: Flow chart of the manufacturing process for CSN CMH-I ^L / CD90 ^H for therapeutic use - manufacturing process with conditional analysis. Organizational chart allowing a double control conditioning the industrialization of therapeutic units and their use. Unlike the manufacturing process assisted by analysis matrix (Figure 16), cell characterization is based on a conditional analysis: if the population of CSN analyzed is heterogeneous, the proportion of the CMH-I ^L / CD90 subpopulation ^H can be assessed using an analysis matrix. If the population is homogeneous, the population must meet the threshold conditions.

Figure 18: Representative examples of establishing CSN analysis matrices with the CMH-I and CD90 double marking tool.

CSNs are isolated from canine placenta or thawed in early passage and amplified in several passages. The cells are analyzed by double labeling CMH-I and CD90. Here the two examples show an individualization of the CMH-I ^L / CD90 ^H and CMH-I ^H / CD90 ^L subpopulations during the amplification. The two examples made it possible to establish an analysis matrix for the quantification of these two types of CSN subpopulations. CSNs moving towards a CMH-I ^L / CD90 ^H phenotype are considered to be industrializable (CSN-2).

EXAMPLES

EXAMPLE A: Method for Obtaining a CSN Population

1. Isolation of CNS from canine placenta

The canine extra embryonic appendages (placenta, umbilical cord) are removed aseptically during cesarean sections performed in pregnant bitches at term. As soon as the newborn puppy is removed from the amniotic sac and put in safety, the extra embryonic tissue is immediately transferred to a transport box containing a phosphate-buffered saline solution from Dulbecco (D-PBS) to be sent to the laboratory. The processing of the extra embryonic tissue is carried out at most within 48 hours of the sample. All of the tissue processing steps are carried out in a controlled environment, under a microbiological safety station (PSM).

The tissue is transferred to a 100 cm 2 petri dish and the residual amniotic membrane is removed mechanically by dissection. The placenta is placed embryonic face against the plastic surface of the box and the uteroverdin present on the face of maternal origin is separated from the placenta by scraping the tissue. The placenta is rinsed 3 to 5 times in successive baths of D-PBS. The blood vessels and umbilical cord are then removed mechanically from the placenta. The placental tissue is dissected into fragments of approximately 10-20 mm ² and then subjected to an enzymatic digestion by incubating the tissue fragments in a solution composed of DMEM (modified Eagle's medium from Dulbecco) containing 0.5-4 mg / ml of type I collagenase, and more specifically a concentration of 1 mg / ml type I collagenase. The enzymatic digestion takes place at 37 ° C. for 1 h but digestion between 30 min and 16 h can be carried out by reducing the temperature of incubation (room temperature (18-22 ° C or 4 ° C). At the end of digestion, the enzymatic activity is stopped by dilution, by adding DMEM containing at least 10% fetal calf serum (S VF) in amount equivalent to the enzymatic digestion solution, the solution is then filtered through a 70-100 mhi sieve, the recovered cells are centrifuged at 800 g for 10 min, the cell pellet containing the neonatal stromal cells is rinsed with DMEM and no. uug centrifuged at 800 g for 10 min. The cell pellet is taken up in culture medium consisting of

DMEM, 10% S VF, 2 mM glutamine and 0 to 20 ng / ml of fibroblast growth factor (FGF). The cells are counted and seeded in culture dishes at a density of between 10 ⁴ and 2.10 ⁴ cells / cm ² . The cells are then cultured in the culture medium described above in a controlled atmosphere at 37 ° C. and containing 5% of CO ₂ . The medium is changed after 48 hours and then every 2-3 days. The cells are passed when the confluence reaches 70-80%.

2. Isolation of CSNs from Equine Umbilical Cord Blood

Equine umbilical cord blood or placental blood (SPL) is recovered during foaling. It is performed as aseptically as possible by puncturing umbilical cord blood at the umbilical vein using a needle connected to a blood collection bag. The SPL bags are sent to the laboratory under controlled temperature conditions (4 -12 ° C) and must be treated within 48 hours of collection. All of the sample processing steps are carried out in a controlled environment, under a microbiological safety station (PSM).

The SPL is transferred to a sterile container and diluted to half with D-PBS or any other physiological solution. The SPL thus diluted is deposited on a Ficoll solution (1.077 g / ml) contained in a tube, at the rate of 2 volumes of blood diluted to half for 1 volume of Ficoll. The tubes are centrifuged for 20-45 min at 700-1000 g without any step of braking. The SPL is then separated by density gradient and a cell ring is formed at the interphase between the diluted plasma and the Ficoll. The cell ring is recovered by aspiration and washed with D-PBS in a final volume of 50 ml. The cells are then centrifuged between 300-500 g for 5 to 10 min. An erythrocyte lysis step can be performed by incubating the cell pellet with erythrocyte lysis buffer for a few minutes (3-10 min). Make up to 50 ml with D-PBS. The cells are centrifuged between 300-500 g for 5 to 10 min. The cell pellet containing the neonatal stromal cells is taken up in DMEM containing 2 mM of glutamine, 10% of FCS and 0-20 ng / ml of FGF (amplification medium). The cells are counted and seeded in culture dishes at a density of between 10 ⁵ and 2, 5.10 ⁵ cells / cm ² . The cells are then cultured in an amplification medium in a controlled atmosphere at 37 ° C. and containing 5% of CO ₂ . The medium is changed after 48 hours and then every 2-3 days. The cells are passed after the emergence of fibroblastic colonies after 10-15 days.

3. Cell passage and amplification

At sub-confluence, the cells undergo cell passage and optionally, an amplification procedure. The CSNs are rinsed with D-PBS and treated with 0.05% trypsin-EDTA for 2-5 min at 37 ° C. This makes it possible to detach the cells and form a population of isolated cells. The cells are then taken up with amplification medium consisting of DMEM, 10% of FCS, 2 mM glutamine and from 0 to 20 ng / ml of fibroblast growth factor (FGF) and centrifuged between 300-500 g for 5 to 10 min. The CSNs are taken up in amplification medium, and counted by manual counting (tryan blue and Malassez cells) or using a cytometer. They are then seeded at a height of 1500 to 5000 cells / cm ² and cultivated on a plastic cell culture support in amplification medium and in a controlled atmosphere at 37 ° C. and containing 5% of CO ₂ . During the amplification process, cells can undergo between 0 and 15 cell passages.

4. Freezing and cryopreservation of CSNs

At the end of the first or second cell passage (P1-P2), the cells can be cryopreserved in cellular seed units. To do this, after counting, the CSNs are centrifuged between 300-500 g for 5 to 10 min and the cell pellet is taken up in freezing medium composed either of enriched DMEM medium. with 5-20% of FCS and 5-10% (vol: vol) of DMSO or in a commercial cryopreservation medium, containing or not a DMSO fraction. The cell concentration is between 1.10 ⁶ and 15.10 ⁶ cells per ml of freezing medium. The cells are frozen under conditions of controlled temperature reduction, for example using a CoolCell® Cell Freezing Containers (BioCision) and following the freezing procedure as described by the manufacturer. The cells are then transferred for negative cold storage at temperatures between -70 ° C and -196 ° C.

The seed cell units can be used to generate therapeutic cell units. The cellular seed units are thawed at 37 ° C for 3-6 min and amplified in vitro. The cells are seeded in culture medium at a density of 1500-3000 cells / cm2. The cells are amplified by successive passage in vitro. When a significant number of cells is produced (for example> 150 × 10 ⁶ cells), the cells are frozen according to the protocol described above. The cells are distributed in hermetically sealed sealable bottles at a rate of 1.10 ⁶ - 15.10 ⁶ cells / ml in freezing medium free of product of animal origin (such as the cryopreservation medium Recovery ™ Cell culture freezing medium (Thermo Fisher) , Cryostem ™ freezing medium (Biological Industries). The vials are lowered in temperature using a controlled temperature lowering protocol, at -l ° C / min to -80 ° C. The vials are then transferred to -80 ° C for storage Once obtained, the CSN population is characterized on the one hand by its structural characteristics (presence / absence of markers) and on the other hand by its functional characteristics (proliferation, differentiation etc.).

EXAMPLE B: Structural characterization of the CSN population

1. Cytometric analysis of CSNs

Cytometric analysis aims to determine the presence of membrane markers on the surface of CSNs, in particular CD1 lb, CD14, CD31, CD34, CD45, HLA-DR and more precisely to determine the expression levels of MHC-I and CD90. , through the use of a panel of antibodies specific to each marker.

After isolation of the cells and during the amplification period, CSNs on passage P2 to P7 cultured in a T75 are rinsed with 10 ml of D-PBS. 5 ml of trypsin / EDTA are then added and the cells are incubated for 2 min at 37 ° C. The cells are peeled off and collected in a 15 ml tube. The volume is adjusted to 15 ml by adding amplification medium. The cells are centrifuged for 10 min at 300-500 g. The supernatant is aspirated then the cell pellet is taken up in 2 ml of amplification medium. 40 mΐ of the suspension are diluted with 40 mΐ of trypan blue. The dilution is then counted by depositing on a counting slide and acquisition by a cell counter of the Luna type.

Generally between 10 ⁵ -5.10 ⁵ cells are recovered for cytometric analysis. In particular, 3 samples of 2.10 ⁵ cells are transferred into 1.5 ml eppendorfs. The cells are taken up in 1 ml of D-PBS and centrifuged at 500 g for 5 min. A second washing is carried out under the same experimental conditions. After removal of the supernatant, the cells of the three tubes are respectively taken up in 1 volume of 30-100 mΐ, in particular 50 mΐ, of labeling buffer. This buffer is composed of D-PBS and 0.5-1% (v / v) bovine serum albumin (BSA) or 0.5-2% (v / v) fetal calf serum. 2.5 mΐ of a primary antibody coupled or not coupled to a fluorochrome and specifically targeting the membrane marker of interest is added to the 50 mΐ of labeling buffer.

The optimal concentration of antibody used for labeling must be determined beforehand by a person skilled in the art. The incubation required for labeling must also be determined by a person skilled in the art and between 15 min and 10 h at 4 ° C. protected from light. In particular, the cells are incubated for 20 min at 4 ° C. protected from light.

Labeling with a secondary antibody targeting the primary antibody can be carried out, after washing with D-PBS, in the case where the primary antibody is not directly coupled to a fluorochrome. Thus, in the case where the primary antibody is not coupled to a fluorochrome, 1 ml of D-PBS is added and the cells are centrifuged for 5 min at 500 g. The supernatant is removed and 50 mΐ of labeling buffer (D-PBS / 2% BSA) comprising 1 mΐ of secondary antibody solution targeting the primary antibody and labeled with a fluorochrome phycoerythrin (PE) is added. The cells are incubated for 20 min at 4 ° C. protected from light. Subsequently 1 ml of labeling buffer is added and the cells are centrifuged for 5 min at 500 g. The cells are taken up in 200 ml of labeling buffer. Isotypic controls adapted to each labeling must be used as a negative control Following the incubation with the antibodies, the cells are washed in D-PBS, centrifuged 5-10 min at 500 g and taken up in a volume of 100 to 250 mΐ of marking buffer for analysis with a flow cytometer (Accuri C6, BD Biosciences). 2. Selection based on expression of CMH- and / or CD90

at. Cytometric analysis on validated antibody with thresholds of

defined positivity / negativity

The cytometric analysis is carried out so as to guarantee a signal resulting from the fluorescence of the antibodies coupled or not with a fluorochrome which are potentially fixed specifically on the epitopes of interest (belonging to CD90 or to MHC-I). To do this, a person skilled in the art will have to correctly and meticulously adjust the photomultipliers and the fluorescence compensations. Furthermore, careful selection of the cell population to be analyzed must be carried out. On the other hand, it must use unmarked control samples, use control samples marked with adequate total IgG coupled to the fluorochromes of interest and this in order to guarantee that the signal measured on the analyzed samples takes both into account, l auto fluorescence of cells and possible non-specific binding of antibodies. Potential treatment of cells with Fc receptor blockers may also be considered.

In order to determine the thresholds of positivity / negativity of the cells, the respective good isotypic controls of each antibody coupled to their fluorochrome must be carefully selected.

In Figure 1, four CSN populations were analyzed by cytometry. An analysis of the unmarked cells is performed first. An analysis window based on the FSC-A / SSC-A analysis parameters is defined to select the events corresponding to the cells and to eliminate cellular debris. Subsequently, an analysis window based on the FSC-H / FSC-A analysis parameters is defined for the analysis of single cells (elimination of cell doublets) (Figure 1 A).

Fe tube marked by a primary isotype is analyzed according to the previous parameters. Fe PE labeling is displayed on the appropriate fluorescence channel corresponding to the fluorescence of interest of the cytometer (in FIG. 1B, the channel used is FF2). The analysis is carried out in the form of a 2D representation (histogram with 2 parameters) based on the analysis of the fluorescence from the fluorochrome used (FF: 2 for PE) and the auto fluorescence of the cells using a fluorescence channel with an excitation / emission spectrum not used in the analysis (FF-4) (histogram FF / counting of events). Fe positivity threshold is placed so that less than 0.5% and more than 0.1% of the cells are considered positive. This same process is carried out for each of the markers CMH-I (Figure 1C) and CD90 (Figure 1D and 1E). Fes 4 analyzed samples have variable but homogeneous expressions of the MHC-I (Figure 1C) while the analysis of CD90 highlights two distinct populations within the CSN samples (Figure 1D), for this latter marking it is thus possible to determine the proportion of cells expressing or not CD90. The CSN-1 and CSN-2 populations meet the selection criteria for an expression of MHC-I of less than 10% and an expression of CD90 of more than 80%. Therefore CSN-1 and CSN-2 are considered as MHC-I ^L / CD90 ^H while the populations CSN-3 and CSN-4 are considered as MHC-I ^H / CD90 ^l .

b. Size variations / auto fluorescence and 2D representation

It is important to keep all of these parameters fixed for the analysis of CSN populations of interest in order to determine the proportion of CSN expressing or not the markers.

Variations in the size of CSN populations may be observable. Thus, the selections of the analysis windows or "spoils" of the populations to be analyzed must not vary between samples. However, in the case where a heterogeneous population in size obstructs the cytometric analysis, an in silico separation of the different homogeneous subpopulations in size can be considered. In the latter case, new positivity / negativity thresholds are to be determined for each sub-population by the skilled person.

It is possible that in parallel with a variation in size, a variation in F auto fluorescence of the cells may be observable. In the latter case, a mode of representation of the results on two acquisition channels (ex: FL-2 / FL-4) is necessary in order to finely define the variations in expression of the markers between the different sub-populations emitting levels variable auto fluorescence. Here again, the determination of new positivity / negativity thresholds may be carried out by a person skilled in the art. It should nevertheless be noted that in the case where the CSN population analyzed has a morphology or autofluorescence too far from the original populations, the latter can be considered as non-compliant and therefore excluded.

vs. Example of determining maximum relative MFI thresholds for

MHC-I with the use of two different secondary antibodies

By maximum relative MFI threshold (rMFI), we mean the limit relative MFI value allowing to keep, for the industrialization procedure, all CSN strains having an rMFI lower than this value. Based on the configuration and analysis presented in section B. 2 a) above, several samples of CSN are analyzed.

Seven populations of CSN from seven different placentas were analyzed from a proliferative point of view. Two of them allowed an amplification greater than 20 cell doublings. These cells were analyzed between P3 and P4 according to the procedure described in example B, part 1).

As examples, we detail below the determination of two maximum rMFI thresholds for two different immunostaining strategies (Figure 2A and Figure 2B). The characteristics of the antibodies used for these two strategies are listed in table 2 below.

Table 2: Characteristics of the antibodies used

The isotype used is a primary e-COL2002 / COLIS205C IgG2a mouse isotype not coupled to a fluorochrome (Monoclonal Antibody Center Washington State University). A canine anti-MHC-I antibody of the mouse type DG-BOV2001 / DG-H58A IgG2a was used for the primary labeling of MHC-I (Monocloal Antibody Center Washington State University).

Two types of secondary antibodies rabbit F (ab ') 2 anti IgG mouse STAR12A (AbSerotec) and goat t F (ab') 2 anti mouse secondary IgG (eBioscience) coupled to a fluorochrome type PE (Figure 2A) and APC respectively (Figure 2B) were used for this study and made it possible to determine two maximum rMFI thresholds specific to each secondary antibody.

In accordance with the part Example B. 2) a) of the present application, the cells marked by the isotype or by the MHC-I are analyzed with the C6 Accuri cytometer. The channels used for the analysis of the fluorescence emitted correspond to the FL-2 channel for the rabbit F (ab ') 2 anti mouse IgG STAR12A antibody (AbSerotec) and FL-4 for the goat F (ab') 2 anti antibody secondary IgG mouse (eBioscience). For each antibody and each strain of CSN (labeled with G isotype and with MHC-I), the values of the MFIs are extracted. The rMFIs of each sample labeled with MHC-I are calculated by normalizing the MFI values of the labeled cells relative to those of the cells incubated with F isotype (labeled MFI / isotype MFI).

Analysis of the expression of the MHC-I indicates variable expressions of this marker between the different samples. The proposed analytical method consists in normalizing the mean fluorescence values (MFI; Mean Fluorescence Intensity) of the cells labeled with the antibody of interest (here MFI FL2 (CMH-I-PE)) with the MFI of the cells labeled with the isotype coupled to the same fluorochrome (here MFI FL2 _(isotype-PE) ) · A ratio (rMFI) is thus calculated MFI FL2 (CMH-I-PE) / MFI FL2 _(isot y _pe -PE ₎ .

For each secondary antibody used:

the highest rMFI value of the strains meeting the criteria of minimum doubling number, positive chondrogenesis, negative osteogenesis and positive immunomodulation is taken for the calculation of the maximum threshold of rMFI;

- and the lowest rMFI value of the strains whose doubling number is less than 20 is also retained.

Thus, for the rabbit F (ab ') 2 anti IgG STAR12A mouse (AbSerotec) antibody labeled PE the average of these two rMFI values is calculated and makes it possible to determine a maximum rMFI threshold of 2.5 (FIG. 2A). For the goat antibody F (ab ') 2 anti mouse IgG secondary (eBioscience) marked APC, the average of these two rMFI values is calculated and makes it possible to determine a maximum rMFI threshold of 10 (Figure 2B).

d. Example of determining acceptance thresholds for CD90

The same approach described in section 2) c). can be performed for the determination of the minimum rMFI threshold for CD90 making it possible to exclude cells having a too low CD90 expression level. The analysis of CD90 in CSN, it is expressed in a substantially heterogeneous way in the different populations of CSN. Unlike CMH-I, the loss of expression of CD90 materializes by the emergence of a distinct subpopulation. Two levels of CD90 expression are thus observable, a weak expression (CD90 ^L ) and a strong expression (CD90 ^H ). As a result, the two normal populations highlighted do not always allow the possibility of determining a single value of MFI. e. Example of determining acceptance thresholds for CMH-I and CD90 by a strategy of double labeling on a population of heterogeneous CSNs.

In order to limit the constraints linked to heterogeneous populations for the determination of thresholds, the same approach described in section 2) c). can be performed for the determination of the minimum rMFI threshold for CD90 and maximum for MHC-I and this from isolated heterogeneous (mixed) populations. Among 14 CSN populations analyzed by CD90 and CMH-I double labeling during cell amplification, only one population of CSN did not pass through a heterogeneous population stage with regard to CD90 and MHC-I. On the 13 others, a mixed population stage (ie represented by the coexistence of two populations CMH-I ^H / CD90 ^L and CMH-I ^L / CD90 ^h ) could be observed between Pl and P4. These two sub-populations have always shown differences in expression of the two markers, highlighting a population of interest MHC-I ^L / CD90 ^H and another population MHC-I ^H / CD90 ^l . Analysis of the rMFIs of the two respective sub-populations CMH-I ^H / CD90 ^L and CMH-I ^L / CD90 ^H makes it possible to establish discrimination thresholds in order to identify the population of interest CMH-I ^L / CD90 ^H ( Figure 3). Regarding CMH-I, the threshold for identifying a CMH-I ^L / CD90 ^{H population} and minimizing false positives is a maximum rMFI of 15. For CD90, the minimum rMFI is 20.

EXAMPLE C: Biological characterization of the CSN population

1. Evaluation of the proliferative activity of the CSNs

The proliferation of CSNs is evaluated for 7 to 8 consecutive cell passages (if feasible). At each cell passage (once a week; ie every 6 to 8 days), the cells are detached from their culture support using 0.5% trypsin / EDTA for 2 -3 minutes. Culture medium is added and the cells are centrifuged for 5 min / 300 g. The cell pellet is taken up in a defined volume of culture medium and the cells are counted by trypan blue exclusion technique using an electronic counter. The number of doublings at each cell passage is calculated according to the following formula: Nb of doublings = LOG (Nf / Ni) / LOG (2) (Nf: number of final cells and Ni: number of initial cells). The total number of cell doublings is equal to the sum of the number of doublings accumulated during each cell passage. Cellular amplification is stopped as soon as the number of doublings is less than 1, which means that the cell population is no longer able to double. The proliferative activity of a population of MHC-I ^L / CD90 ^H cells is compared to that of a population of MHC-I ^H / CD90 ^L cells (FIG. 4).

A CSN cell population is considered acceptable for industrialization if the total number of consecutive cell doublings is greater than or equal to 20. As shown in Table 3, cells of phenotype MHC-I ^L / CD90 ^H have a number total consecutive cell doubling of cells greater than 20, unlike CMH-I ^H / CD90 ^l cells. This clearly demonstrates the CSNs of phenotype CMH-I ^L / CD90 ^H has a proliferative activity much superior to cells CMH-I ^H / CD90 ^l , which thus allows them to be used for industrialization unlike cells CMH-I ^H / CD90 ^l .

2. Chondro enique differentiation

The capacity for chondrogenic differentiation of the CSNs is verified by incubation of the cells in a differentiation medium specific for this differentiation pathway.

For this, the CSNs are detached from their plastic support by trypsinization and counted. The cells are then used to form micromasses by gravitation in a drop of amplification medium. To do this, a solution of approximately 7.10 ⁶ cells / ml of amplification medium is then prepared. 35 mΐ of this correctly homogenized solution are then deposited in the form of a drop on an untreated and uncoated plastic surface. This surface is then returned to 24h incubation in a humid atmosphere at 37 ° C and 5% C0 ₂ . For example, the lid of a Petri dish turned over on its base containing D-PBS can be used as a culture support.

After 24 hours of incubation, a CSN micromass formed at the base of the drop. The micromass is collected and transferred to a 6-well plate so as to deposit 5 micromasses per well. 2 ml of chondrocyte differentiation medium are then added per well in order to induce the chondrogenic differentiation of the CSNs. This medium consists of DMEM 4.5 g / l of glucose, isulin-selenium-transferin IX, dexamethasone 0.1 mM, sodium pyruvate 1 mM, 50 μg / ml of ascorbic-2-phosphate acid , 40 pg / ml of L-Proline, 10 ng / ml of TGF-b3 (or a combination of 10 ng / ml of TGF- bΐ and 50 ng / ml of BMP-2). Chondrogenic differentiation is carried out within 7 days by changing the differentiation medium every 2-3 days. Acquisition under the microscope photonics is carried out at the end of the culture by a Nikon TS2 x4 magnification coupled to a TS2-P-CF camera.

Three populations of CSN were analyzed: CNS-1, CSN-2 and CSN-3. The CSN-1 population has the MHC-I ^L / CD90 ^H phenotype while the CSN-2 and CSN-3 populations have the MHC-I ^H / CD90 ^L phenotype.

At the end of the culture, the micromasses observed under the microscope show a substantial difference in size (Figure 5). The surface occupied on the image by micromasses is analyzed under ImageJ and expressed in pixels ² . The CSN CMH-I ^L / CD90 ^H form a larger chondrogenic micromass than the CSN CMH-I ^H / CD90 ^L (Figure 5A and Figure 5B).

The medium is subsequently removed from the wells and the neo-tissue and used for RNA or protein extraction in order to analyze the expression of markers specific for the chondrogenic lineage such as collagen type II, aggrecan, COMP, SOX9.

The total RNAs are extracted by grinding the micromasses using a plastic pestle in the presence of Trizol Reagent (Sigma). The rest of the extraction procedure is carried out under the manufacturer's conditions. RNAs are back transcribed using PrimeScript Reverse Transcriptase (Clonetech) according to the manufacturer's conditions. The RTqPCR analysis is performed using an Mx3000p thermal cycler as well as MxPRO software (Stratagene). The results are expressed as the rate of expression of the target genes relative to a housekeeping gene (of the Gapdh type) using the 2-AACT method. The expression of the Col2al gene was analyzed by RTqPCR and normalized by the expression of the Gapdh gene. This expression of the Col2al gene was followed in the micromasses obtained for each cell population (CSN-1, CSN-2 and CSN-3) after 7 days of chondrogenic differentiation (treated) compared to this same cell population which has not undergone of chondrogenic differentiation (Ctrl). The data are expressed in relative expression of Col2al with respect to the control (Figure 5C).

We consider acceptable an expression value of Col2al relative to undifferentiated cells grown on plastic support greater than 100.

The expression of these markers makes it possible to conclude that there is a potential for chondrogenic differentiation of the CSNs.

FIG. 5D demonstrates that the expression of the Col2al gene in the population CSN-1 of phenotype MHC-I ^L / CD90 ^H is higher than in the populations CSN-2 and CSN-3 which are of phenotype MHC-I ^H / CD90 ^l . This makes it possible to conclude that there is a greater potential for chondrogenic differentiation in CSN CMH-I ^L / CD90 ^h . On the analysis of 8 populations of CSN, the results showed a difference in Col2al expression between the CSN CMH-I ^H / CD90 ^L and CMH-I ^L / CD90 ^H (Figure 5D).

3. Osteogenic differentiation

The low osteogenic differentiation capacity of the CSNs is verified by incubation of the cells in a differentiation medium specific for this differentiation pathway.

For this, the CSNs are detached from their plastic support by trypsinization and counted. The CSNs are seeded at a density of between 1500 and 5000 cells / cm ² in a 6-well plate in amplification medium under a controlled atmosphere at 37 ° C. and containing 5% of CO ₂ . When the cells reach 50-75% confluence, the proliferation medium is removed and replaced by osteogenic differentiation medium composed of DMEM, 10% of SVF, 2 mM glutamine, 0.1 mM dexamethasone (Sigma), 50 mM acid. ascorbic 2-phosphate (Sigma) and 10 mM b-glycerophosphate (Sigma). The medium is renewed 2 times / week, for a period between 10 and 15 days.

At the end of the differentiation process, the wells are washed with D-PBS and the cells are fixed with, for example, a neutral buffered formalin solution 10% for at least 1 hour. Staining with a 1% Alizarin red solution (weight / volume) is carried out to demonstrate the presence of calcium deposits. The wells are then rinsed in H ₂ 0 and the coloration is analyzed under a microscope.

The absence of calcium deposits allows us to conclude that there is no osteogenic differentiation potential.

4. Evaluation of the immunomodulatory potential of the CSNs by the expression of PGE2

The potential of CSNs to express molecules exerting an immunomodulatory effect is evaluated by assaying the molecules secreted by CSNs of phenotype CMH-I ^L / CD90 ^H in basal condition or after stimulation with a proinflammatory cytokine.

For this, the CSN CMH-I ^L / CD90 ^H seeded at 5.10 ⁴ cells / cm ² are cultured in proliferation medium as described above for 72 h or in medium supplemented with 5 ng / ml of gamma interferon. At the end of this incubation, the culture medium is centrifuged for 10 min / 500 g. The supernatant is frozen at -80 ° C. The analysis of the factors of interest is carried out by ELISA test by following the instructions specific to each kit. The prostaglandin E2 (PGE2) assay is carried out using the KGE004B kit (R&D System). The results in Ligure 6 show an increase in the expression of PGE2 when the CSNs are treated with a proinflammatory cytokine such as ILN, which demonstrates the potential of CSNs to express molecules, in particular PGE2, which can exert an immunomodulatory effect.

5. Evaluation of the immunomodulatory potential of CSNs by their antiproliferative effect on lymphocytes

The capacity of CSNs to inhibit the proliferation of lymphocytes in vitro is evaluated by co-cultivating CSNs of phenotype MHC-I ^L / CD90 ^H with mononuclear blood cells (PBMCs) in the presence of a mitogenic agent.

For this, the PBMCs are isolated from a blood sample taken from a donor dog or from a donor horse on a Ficoll gradient. The PBMCs are then incubated with a fluorescent dye (CellTrace CFSE, Thermo Fisher) which makes it possible to measure cell proliferation over several generations. 0.2.10 ⁶ PBMCs labeled with Celltrace are added to the wells of a 96-well plate in which the CSNs were seeded the day before at the concentration (2.10 ⁴ CSN / well); thus obtaining a CSN: PBMC ratio equivalent to 1: 10. The CSNs are treated with mitomycin (10 μg / ml for 1.5-2 hours at 37 ° C.) and rinsed 3 times in culture medium before adding the PBMCs. The lymphocyte proliferation medium is added (RPMI, 10% FCS, 2 mM glutamine, 10 mM hepes, 50 mM b-mercaptoethanol, 5 μg / ml concanavalin A). The PBMC / CSN co-cultures and the control cultures (PBMC without CSN) are incubated for 4 days in an incubator at 37 ° C. At the end of the culture, the non-adherent cells are recovered from the wells, centrifuged and washed with D-PBS. The cells are then labeled with an anti-CD3 antibody coupled to a fluorochrome FITC for 30 min at 4 ° C. The cells are then washed with D-PBS before analysis with a flow cytometer (Accuri C6, BD Biosciences). Cytometric analysis consists of evaluating the Celltrace signal in the viable CD3 + population. A proliferation index (PI) is calculated using analysis software, such as Modfït® for example. The proliferation index of PBMCs cultured in the presence of mitogenic agent without CSN (IP Ctrl) is arbitrarily fixed at 1. The proliferation index of PBMCs cultured in the presence of mitogenic agent and CSN at a ratio of 1: 10 (IP 1:10) is normalized to (IP Ctrl). The CSN line is considered to exert significant antiproliferative activity if the ratio (IP 1: 10) / (IP Ctrl) is less than or equal to 0.5. The significant decrease in the proliferation index in the cocultures with CSNs in FIG. 7 demonstrates that the CSNs of phenotype MHC-I ^L / CD90 ^H exert an antiproliferative activity on the lymphocytes. The CSNs therefore have an immunomodulatory potential in vitro, and in this case an immunosuppressive potential. EXAMPLE D: Evaluation of the biological properties of cryopreserved CSNs

The biological activity (viability, proliferative activity, immunomodulatory potential) of CSNs of phenotype CMH-I ^L / CD90 ^H is evaluated in vitro and compared with the properties of CSNs maintained in culture.

The CSNs cryopreserved at -80 ° C for several months are thawed at room temperature for 8-10 minutes. The cell suspension in its cryoprotective medium is transferred into a 15 ml Falcon tube. An aliquot (50m1) is taken to carry out a Trypan blue marking. The sample thus prepared is analyzed using an electronic counter which estimates the cell viability by making the ratio of the number of cells labeled with Trypan blue / total number of cells detected. Alternatively, an aliquot of 50m1 of cell suspension is taken and mixed with 50m1 of propidium iodide solution (IP; 10pg / ml). The solution thus prepared is immediately analyzed on a flow cytometer. The cells showing a signal on the IP detection channel correspond to dead cells.

The capacity of CSN CMH-I ^L / CD90 ^H to proliferate in vitro post-thawing is evaluated by inoculating a defined quantity of cells on a culture support in proliferation medium for 7 days. The proliferative activity of the CSNs is evaluated according to the method described above (paragraph 1).

The immunomodulatory potential of cryopreserved CSNs is evaluated according to the method described in paragraph 5 of the examples.

The results of FIG. 8 show that the cell viability of the CSNs of phenotype CMH-I ^L / CD90 ^H is greater than 80% up to 12 months of storage at -80 ° C (Figure 8A). The proliferative activity of cryopreserved CSNs at -80 ° C. is not significantly modified compared to CSNs maintained in culture, as indicated by the number of doublings and the time of cell doubling which are similar whatever the time of storage at -80 ° C (Figure 8B). The immunomodulatory activity of cryopreserved CSNs is maintained, as shown by the results of the lymphocyte proliferation inhibition test in vitro (Figure 8C and Figure 8D). EXAMPLE E: In vivo evaluation of the therapeutic effect of an injection of cryopreserved CSNs ready for use for the management of osteoarthritis in dogs

Clinical case 1 (Dog 1): 10-year-old English bulldog with dysplasia of both hips as well as rupture of the anterior cruciate ligament leading to marked chronic lameness and almost permanent pain in the hind limbs, characteristics of osteoarthritis . Treatment by intra-articular injection of cryopreserved CSN from a canine placenta (individual different from the individual treated), is prescribed for each joint. Three units of 1.10 ⁷ CSN cryopreserved are thawed 24 hours before administration. The treatments are sent to the veterinary clinic by a temperature-controlled transporter (4 -12 ° C). Each of the three CSN units is directly injected, respectively, into each of the hips and into the stifle, without prior washing of the cells. The evolution of the animal is monitored by a questionnaire developed and validated to assess the mobility of dogs (LO AD; Liverpool Osteoarthritis In Dog). This document is given to the owners of the animal to evaluate in a semi-quantitative way the evolution of the lameness and the comfort of the animal thanks to 13 questions noted from 0 to 4. The score thus varies between 0 and 52, 0 corresponding to the score of a healthy dog and 52 corresponding to the highest pain and discomfort score.

Before treatment, the score is evaluated at 41/52, which corresponds to joint pain / gene which can be considered extreme. Three months after the injection, the score assigned by the owners is evaluated at 28/52, which corresponds to a positive change in locomotion of the animal of 32%.

Clinical case 2 (Dog 2): 10-year-old Yorkshire with marked lameness in the elbow and diagnosed with elbow dysplasia with fragmentation of the medial coronoid process associated with arthritis lesions. Treatment by intra-articular injection of cryopreserved CSN from a canine placenta (individual different from the individual treated), is prescribed for the elbow. A unit of 1.10 ⁷ CSN cryopreserved is thawed 24 hours before administration. The treatment is transported to the veterinary clinic by a temperature-controlled transporter (4 -12 ° C). The CSN unit is injected directly into the elbow, without first washing the cells. The effectiveness of the treatment is evaluated 2 months post-treatment by means of a clinical evaluation carried out by the veterinarian and by the LO AD questionnaire completed by the owners. The clinical evaluation of the animal is to assign a score based on an examination of the mobility of the animal. The following criteria are evaluated: lameness (1-5), pain on palpation (1-3), swelling of the joint (1-3), crackling (1-3); or a total score of 14 (14 corresponding to the most critical condition).

Before treatment with CSN, the clinical score assigned to the animal is 10/14 (lameness at 3/5 (moderate); pain on palpation at 3/3 (severe), swelling of the joint at 2/3 (moderate) and a crackling 2/3 (moderate) The owner score before treatment is evaluated at 34/52.

The results at 2 months post-injection show a clinical score of 6/14; or a clinical improvement of 59% and a proprietary score of 20/52; or a 41% improvement in mobility.

Figure 9 represents the evolution of the LOAD score of each animal before treatment and post-administration of cryopreserved CSN (dog H: 3 months post-injection / dog D: 2 months post-injection)

EXAMPLE F: Evaluation of the Evolution of the Mobility of Dogs Suffering from Osteoarthritis Following the Administration of a Preparation of Cryopreserved CSNs Ready to Use

Fifteen dogs suffering from articular osteoarthritis (knee, elbow, hip, or stifle) were recruited for an intra-articular injection of 1.10 ⁷ CSN cryopreserved / joint. These CSNs come from a canine placenta (individual different from the individual treated). The product is thawed 24 hours before administration, transported to the clinic by a transporter under controlled temperature (4 -12 ° C) and injected without washing the cells.

Six months post-injection, a questionnaire is given to veterinarians to assess the effectiveness of the treatment. A score of 1 to 3 is assigned corresponding to a very satisfactory development (1); a satisfactory development (2); an unsatisfactory development (3). The evolution of the animal's mobility is evaluated as satisfactory in 87% of the cases (80% with an evolution considered very satisfactory); 13% of returns consider an unsatisfactory evolution of the animal following treatment (Figure 10). EXAMPLE G In vivo evaluation of the therapeutic effect of an injection of cryopreserved CSN on canine thrombocytopenia

Clinical case: Dachshund (5kg) aged 8 years is presented in consultation with bruises on the abdomen. The anamnesis of this animal reports 2 episodes of thrombocytopenia at the age of 6 months and 6 years. During these two episodes, corticosteroid therapy with an immunosuppressive dose was necessary, suggesting an autoimmune cause of the disease. Blood tests are done, ruling out an infectious disease. Imaging tests do not identify a neoplastic process. The formula count reveals a platelet count of 76.10 x ³ / mm ³ . The average platelet volume (VMP) is 5.7 fl, close to the low limit (5 to 12 fl). The number of neutrophils is measured at 540 / mm ³ , or 8.8% of the total number of leukocytes (6,000 / mm ³ ). The other constants are normal. In view of the animal's history and the absence of any pathology identified, the diagnostic hypothesis is therefore an idiopathic or primary immune-mediated thrombocytopenia.

A corticosteroid and immunosuppressive treatment is prescribed allowing normalization of the temporary number of platelets, with a relapse at 3 months. An infusion of 1.10 ⁷ CSN cryopreserved is carried out intravenously. These CSNs come from a canine placenta (individual different from the individual treated). The cells are suspended in an infusion of 50 ml of NaCl and are administered slowly over a period of 25 minutes. Monitoring (heart and respiratory rate, rectal temperature, mucosal control) is performed during the infusion. Clinical monitoring is carried out over a period of 3 months with platelet dosages.

During the CSN infusion, the animal shows no clinical manifestations or side effects within 72 hours of the infusion. In the month following administration of the CSNs, corticosteroid and immunosuppressive treatments are gradually reduced. One month post-treatment with CSN, the platelet concentration is evaluated at 200 × 10 ³ / mm ³ . Corticosteroid therapy is suspended. Two months post-treatment, the platelet concentration is evaluated at 255.l0 ³ / mm ³ , 3 months post-treatment, the platelet concentration is evaluated at 460.l0 ³ / mm ³ .

The data show a normalization of platelet concentrations up to 3 months post-injection without taking corticosteroids (Figure 11). EXAMPLE H: In Vivo Evaluation of the Therapeutic Effect of an Injection of Cryopreserved CSNs for the Treatment of Chronic Inflammatory Bowel Disease

Clinical case: Dog breed Shar-pei 4 year old female diagnosed with chronic inflammatory bowel disease in February 2017. The dog has diffuse and moderate duodenal enteritis accompanied by lymphoplasmocytic infiltration. These various lesions cause vomiting, diarrhea and chronic abdominal pain. The veterinarian prescribes increasing doses of cortisone to determine the dose necessary to keep the animal stable. The dose of 2 mg / kg / day is chosen. During the 3 years following the start of cortisone treatment, the owner consults with many veterinarians with the aim of reducing the dose currently prescribed, which may be harmful in the long term. None of the treatments made it possible to reduce the dose and the animal had to remain permanently on corticosteroid treatment.

Treatment with intravenous injection of cryopreserved CSN is prescribed. These CSNs come from a canine placenta (individual different from the individual treated). A unit of 1.10 ⁷ cryopreserved CSN is thawed 24 hours before administration. The treatment is transported to the veterinary clinic by a temperature-controlled transporter (4 -12 ° C). The CSN unit is directly injected via a physiological saline infusion, without prior washing of the cells.

Three days after the administration of the cells, the veterinarian decreases the dose of cortisone by half then after 10 days again decreases the dose by half to obtain a dose of lmg / kg every 2 days, i.e. a reduction in the dose in cortisone by 4. Twenty days after the treatment, the dog remains stable and supports perfectly the decrease in cortisone.

REFERENCES

• Bruder SP, Jaiswal N, Haynesworth SE. Growth kinetics, self-renewal, and the osteogeny potential of purifïed human mesenchymal stem cells during extensive subcultivation and following cryopreservation. J Cell Biochem 1997 Feb; 64 (2): 278-94; • Dominici, MLBK, Le Blanc, K., Mueller, L, et al., Minimal criteria for defïning multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy, 2006, vol. 8, no 4, p. 315-317;

• Phinney, D. G. (2012). Functional heterogeneity of mesenchymal stem cells: implications for cell therapy. Journal of cellular biochemistry, 113 (9), 2806-2812);

• Jacobs, Sandra A., Roobrouck, Valérie D., Verfaillie, Catherine M., et al., Immunological characteristics of human mesenchymal stem cells and multipotent adult progenitor cells. Immunology and cell biology, 2013, vol. 91, no 1, p. 32-39;

• Tessier, L., Bienzle, D., Williams, L. B., & Koch, T. G. (2015). Phenotypic and immunomodulatory properties of equine cord blood-derived mesenchymal stromal cells. PLoS One, 10 (4);

• Lepage, S. L, Lee, O. J., & Koch, T. G. (2019) Equine Cord Blood Mesenchymal Stromal Cells Hâve Greater Differentiation and Similar Immunosuppressive Potential to Cord Tissue Mesenchymal Stromal Cells. Stem cells and development, 28 (3), 227-237;

• Sarugaser, R., Lickorish, D., Baksh, D., Hosseini, M. M., & Davies, J. E. (2005).

Human umbilical cord perivascular (HUCPV) cells: a source of mesenchymal progenitors. Stem cells, 23 (2), 220-229;

• Portmann-lanz, C. Bettina, Schoeberlein, Andreina, Huber, Alexander, et al., Placental mesenchymal stem cells as potential autologous graft for pre-and perinatal neuroregeneration. American journal of obstetrics and gynecology, 2006, vol. 194, no 3, p. 664-673;

• Sibov, TT, Severino, P., Marti, LC, Pavon, LF, Oliveira, DM, Tobo, PR, Campos, AH, Paes, AT, Amaro, E., F Gamarra, L., et al., ( 2012). Mesenchymal stem cells ffom umbilical cord blood: parameters for isolation, characterization and adipogeny differentiation. Cytotechnology 64, 511-521.

Claims

claims

1. Population of neonatal stromal cells (CSN), said population comprising neonatal stromal cells of weak MHC-I phenotype (MHC-I ^L ).

2. Population of neonatal stromal cells according to claim 1, said population comprising neonatal stromal cells of phenotype MHC-I ^L and of phenotype strong CD90 (CD90 ^H ).

3. Population of neonatal stromal cells according to one of claims 1 or 2, characterized in that at least 80% in number, in particular at least 85%, more particularly at least 90% of the cells of said population are MHC-I ^L and characterized in that at least 80% in number, in particular at least 85%, more particularly at least 90% of the cells of said population are CD90 ^H.

4. Population of neonatal stromal cells according to one of claims 1 to 4, characterized in that at least 80% of the cells of said population of CSN have a consecutive cell doubling capacity greater than 20 cumulative doublings.

5. Population of neonatal stromal cells according to one of claims 1 to 5, characterized in that at least 80% of the cells of said CSN population have:

a potential for chondrogenic differentiation, and / or

an immunomodulatory potential.

6. Population of neonatal stromal cells according to one of claims 1 to 5, characterized in that the CSNs come from a neonatal tissue sample, in particular from one or more placentas and / or from one or more umbilical cords , or a sample of neonatal fluid, in particular the blood of one or more umbilical cords.

7. Population of neonatal stromal cells according to one of claims 1 to 6, characterized in that the sample comes from neonatal mammalian tissues or fluids, in particular from dogs, cats, horses, or humans. .

8. Population of neonatal stromal cells for its autologous, allogenic or xenogenic therapeutic use in dogs, cats, horses, or humans, for example for treatment: a. tissue or osteo-articular damage, with or without an inflammatory component

b. degenerative diseases, including osteoarthritis, tendinopathy, tissue fibrosis, Alzheimer's, Parkinson's,

vs. autoimmune, inflammatory and / or infectious diseases, including atopic dermatitis, gingivo stomatitis, thrombocytopenia, epidermolysis bullosa, sepsis, inflammatory bowel disease (IBD);

d. transplant rejection, or

e. tumor diseases.

9. Pharmaceutical composition comprising a population of neonatal stromal cells according to one of claims 1 to 7, and a pharmaceutically acceptable vehicle.

10. The pharmaceutical composition according to claim 9, in which the population of neonatal stromal cells comprises from 1.10 ⁶ to 1.10 ⁸ cells in 0.1 ml to 15 ml of pharmaceutical composition, ie a concentration of between 5.10 ⁴ and 1.10 ⁹ cells / ml. , more particularly from 1.10 ⁶ to 1.10 ⁷ cells in 0.5 to 2 ml, ie a concentration of 5.10 ⁵ to 2.10 ⁷ cells / ml.

11. Pharmaceutical composition according to claim 8 or 9, characterized in that said a pharmaceutically acceptable vehicle is a solution comprising a cryoprotective and characterized in that said composition is in frozen form.

12. Ready-to-use solution for injection comprising a unit dose of 1.10 ⁶ to 1.10 ⁸ of a population of CSN as defined in claims 1 to 7, more particularly 1.10 ⁷ , in solution with a cryoprotector.

13. In vitro method for obtaining a pharmaceutical composition of neonatal stromal cells derived from neonatal tissues, said pharmaceutical composition comprising as active ingredient a population of neonatal stromal cells comprising CSNs of phenotype MHC-I ^L , and optionally of CD90 ^h phenotype, said method comprising: a. the supply of one or more neonatal biological samples comprising CSNs, the biological sample or samples having been previously obtained from one or more individuals,

b. isolation of the CSN population present in the biological sample (s),

14. In vitro method for obtaining a pharmaceutical composition of neonatal stromal cells, according to claim 12, characterized in that the neonatal biological sample (s) supplied in step a., Come from a neonatal tissue sample, in particular of one or more placentas and / or of one or more umbilical cords, or of a sample of neonatal fluid, in particular the blood of one or more umbilical cords and / or characterized in that the biological sample (s) neonates provided in step a. come from mammals, especially dogs, cats, horses or humans

15. In vitro method for obtaining a pharmaceutical composition of neonatal stromal cells according to one of claims 12 to 14, characterized in that said method comprises an amplification step c., Said amplification step comprising 1 to 5 cell passages, in particular 2 to 3 cell passages, and / or said amplification step corresponds to a doubling of the population of CSNs isolated in step b. from 2 to 25 cell doublings, in particular 5 to 15 cell doublings.