Mesenchymal stem cells obtained from wharton's jelly for the treatment of sepsis

Abstract

The invention relates to human mesenchymal stem cells obtained from Wharton's jelly, a process for the preparation thereof, and the therapeutic uses thereof, in particular for the treatment of sepsis. A a specific embodiment of the invention relates to the use of thawed human mesenchymal stem cells (MSC) from Wharton’s jelly, for the manufacture of a medicament for the treatment of sepsis, in particular septic shock, wherein: the expression level of the marker CD44 in said thawed human MSC from Wharton’s jelly is at least 20% lower than the expression level of the marker CD44 in fresh human MSC from Wharton’s jelly; and / or, the expression level of at least one marker selected from CD90, CD73, CD105, 10 CD146, CD166 in said thawed human MSC from Wharton’s jelly is at least 10% lower than the expression level of the same marker in fresh human MSC from Wharton’s jelly.

Description

[0001] Pure Wharton's jelly mesenchymal stem cells for the treatment of sepsis

[0002] SCOPE OF THE INVENTION The present invention relates to human mesenchymal stem cells derived from Wharton's jelly, their method of preparation and their therapeutic uses.

[0003] STATE OF THE ART

[0004] Although often overlooked, septic shock, along with myocardial infarction, is the eleventh leading cause of death worldwide. The leading cause of admission and death in non-coronary intensive care units, its incidence, estimated at 50 to 100 cases per 100,000 inhabitants, has been steadily increasing in recent years (Dombrovskiy et al., 2007). Increased life expectancy, the growing number of multidrug-resistant bacteria, and more frequent diagnosis are contributing factors to this phenomenon.

[0005] The pathophysiology of septic shock is particularly complex (Iskander et al., 2013) and simultaneously involves both pro- and anti-inflammatory cytokines as well as immunoparalysis. Consequently, there is no specific treatment for septic shock due to the inability of conventional pharmaceutical molecules to act simultaneously on both the inflammatory and anti-inflammatory aspects while also stimulating the immune system.

[0006] Numerous studies using murine models of endotoxemia or peritonitis have demonstrated the ability of mesenchymal stem cells (MSCs) to decrease plasma levels of IL-6, IL-1p, IL-12, IL-2, and IL-17 (Chao et al., 2014a; Kim et al., 2014; Luo et al., 2014; Pedrazza et al., 2014) and lower tissue concentrations of TNF-α, IL-6, IL-1p, and IL-12 in the lungs, liver, and intestines (Gonzalez-Rey et al., 2009), or in bronchoalveolar fluid (Mei et al., 2010a). The capacity of MSCs to limit neutrophil migration to tissues contributes to moderating the deleterious effects of inflammation on tissues (Rojas et al., 2014).Studies of histological sections from mice subjected to polymicrobial sepsis by cecal ligation and perforation (CLP) or to endotoxemia sepsis show a reduction in lung and kidney tissue inflammation when treated with mesenchymal stem cells (MSCs) (Krasnodembskaya et al., 2010). Similarly, these mice exhibit significantly better biological markers of organ function, such as amylase, creatinine, bilirubin, aspartate aminotransferase (AST), and alanine aminotransferase (ALT), than mice treated with saline solution (Luo et al., 2014; Pedrazza et al., 2014).

[0007] In addition to their ability to limit tissue damage, MSCs are capable of increasing bacterial clearance during septic shock. Indeed, several teams have demonstrated that the injection of MSCs in septic mice led to a decrease in bacteremia by increasing the phagocytic activity of monocytes, macrophages and neutrophils, but also by the synthesis and secretion by the MSCs themselves of antibacterial peptides such as LL-37 and hepcidin (Alcayaga-Miranda et al, 2015; Devaney et al, 2015; Gonzalez-Rey et al, 2009; Hall et al, 2013; Krasnodembskaya et al, 2010; Mei et al, 2010b).

[0008] Furthermore, MSCs possess intrinsic "homing" properties that give them a susceptibility to migrate, following a chemokine gradient, to damaged organs such as the liver, kidneys, and lungs. This ability to reach damaged tissues is a particularly valuable advantage of MSCs in the context of organ failure pathologies such as septic shock (Wannemuehler et al., 2012).

[0009] Finally, the reduction of tissue damage through the injection of MSCs in murine models of severe sepsis leads to increased survival. Numerous studies highlight a significant decrease in mortality in animals treated with MSCs before or after sepsis (Alcayaga-Miranda et al., 2015; Devaney et al., 2015; Gonzalez-Rey et al., 2009; Krasnodembskaya et al., 2012; Luo et al., 2014; Németh et al., 2009). Thus, through their ability to regulate hyperinflammation and moderate tissue damage, MSCs appear to be able to significantly improve post-septic shock survival.

[0010] Three major sources of mesenchymal stem cells (MSCs) are distinguished: bone marrow (BM), adipose tissue, and Wharton's jelly (GW) from the umbilical cord. Compared to BM MSCs, GW MSCs, obtained simply, non-invasively, and without anesthesia, could democratize MSC donation by eliminating the risks associated with BM harvesting. However, while BM and adipose tissue MSCs have been studied in animal models of septic shock, the potential of Wharton's jelly MSCs for this indication remains poorly documented. To date, only five publications have analyzed the therapeutic potential of GW MSCs in septic shock using a murine model of cutaneous leishmaniasis (CLP) (Chao et al., 2014a; Condor et al., 2016; Wu et al., 2015; Zhao et al., 2014). However, the cells used in these studies were fresh cells.No published studies to date concern thawed MSC-GW in the indication of septic shock, this may be due to general knowledge on the impact of freezing and thawing on the pharmacological and immunomodulatory potential of stem cells (François et al, Cytotherapy. 2012.14(2): 147-52; Chinnadurai et al., 2016; Moll et al, 2016).

[0011] Furthermore, Mezey and Nemeth (2015) described a decrease in the antibacterial properties of MSCs when injected late. Consequently, to date, these technical constraints prevent the use of GW-MSCs in clinical settings for therapeutic treatment.

[0012] Contrary to expectations, the inventors of the present invention demonstrate for the first time that thawed CSM-GW retain their therapeutic potential and can be used in the treatment of sepsis. SUMMARY

[0013] The present invention relates to thawed human mesenchymal stem cells (MSCs) derived from Wharton's jelly for use in the treatment of sepsis, particularly septic shock. In one embodiment, the thawed human MSCs derived from Wharton's jelly of the present invention are characterized in that the expression level of at least one marker selected from CD90, CD73, CD105, CD29, CD44, CD146, CD166, and HLA-ABC is at least 10% lower than the expression level of the same marker in fresh human MSCs derived from Wharton's jelly. In another embodiment, the thawed human MSCs derived from Wharton's jelly of the present invention are characterized in that the expression level of the CD90 marker is at least 10% lower than the expression level of the CD90 marker in fresh human MSCs derived from Wharton's jelly.

[0014] In one embodiment, thawed human MSCs from Wharton's jelly of the present invention are characterized in that said MSCs express at least one protein selected from the group comprising ACTB, ANXA1, CAPZB, LASP1, PRDX2, PRDX3, PSA3, RS12 and SYWC.

[0015] In one embodiment, thawed human MSCs from Wharton's jelly of the present invention are characterized in that said MSCs do not substantially express at least one protein selected from the group comprising ACTS, ALIBI, ANX10, GBB1, GBB2, GPRIN1, DTNA, MIPOl, PSB3 and PSDE.

[0016] In one embodiment, the thawed human MSCs from Wharton's jelly of the present invention are characterized in that said MSCs, under in vitro conditions and / or under non-inflammatory conditions, secrete at least one growth factor selected from BMP-7, IGFBP-1, insulin, FGF-7, NT-4 and VEGF-D.

[0017] In one embodiment, the thawed human MSCs from Wharton's jelly of the present invention are characterized in that said MSCs, under in vivo and / or inflammatory conditions, secrete at least one growth factor selected from BMP-7 and TGFp3.

[0018] In one embodiment, the thawed human MSCs from Wharton's jelly of the present invention are characterized in that said MSCs, under in vivo and / or inflammatory conditions, do not substantially secrete IGFBP-1.

[0019] In one embodiment, thawed human MSCs from Wharton's jelly of the present invention are characterized in that said MSCs secrete at least 1.2 times more VEGF than fresh human MSCs from Wharton's jelly. In another embodiment, thawed human MSCs from Wharton's jelly of the present invention are characterized in that said MSCs induce an increase in serum VEGF concentration in the patient of at least 5% compared to fresh human MSCs from Wharton's jelly.

[0020] In one embodiment, the thawed human MSCs from Wharton's jelly of the present invention are characterized in that said cells are derived from human umbilical cord tissue from a mother meeting at least one of the following criteria: having received oxytocin during delivery, having delivered by induced labor, having delivered at term, not having experienced preeclampsia during pregnancy, whose child did not present neonatal disorders and having been exposed to tobacco smoke during pregnancy.

[0021] In one embodiment, the thawed human MSCs derived from Wharton's jelly of the present invention are characterized in that said MSCs are clinical grade cells.

[0022] In one embodiment, the thawed human MSCs from Wharton's jelly of the present invention are characterized in that said cells are directly from thawing without re-culture after thawing.

[0023] The present invention also relates to a pharmaceutical composition comprising thawed human MSCs from Wharton's jelly according to the present invention and a pharmaceutically acceptable excipient, for use in the treatment of sepsis.

[0024] The present invention also relates to a method for preparing clinical-grade mesenchymal stem cells from Wharton's jelly, comprising the following steps:

[0025] (i) to culture human umbilical cord tissue containing Wharton's jelly in a clinical-grade culture medium for cell adhesion;

[0026] (ii) incubate the adherent cells in a medium containing platelet lysate;

[0027] characterized in that said tissue comes from a mother meeting at least one of the following criteria: having received an administration of oxytocin during delivery, having given birth by induced labor, having given birth at term, not having experienced preeclampsia during pregnancy, whose child did not present neonatal disorders and having been exposed to tobacco smoke during pregnancy.

[0028] DEFINITIONS

[0029] In the present invention, the terms below are defined as follows:

[0030] The term "absence of neonatal disorders" refers to a newborn showing no signs of prematurity, intrauterine growth restriction, or fetal distress. Prematurity is determined by the number of weeks of gestation. Intrauterine growth restriction is determined by birth weight, length at birth, and head circumference. Fetal distress is determined by the arterial pH of cord blood and the Apgar score, defined as follows: A = appearance (color), P = pulse, G = grimace (reflexes to stimuli), A = activity (muscle tone), R = respiration (respiratory effort). The thresholds for these parameters used to determine whether a newborn has no neonatal disorders are well known to professionals.

[0031] A "full-term delivery" is defined as a delivery resulting in the birth of a newborn with a birth weight greater than 3.2 kg, after at least 39.5 weeks of amenorrhea and with the weight of the placenta at the time of delivery being greater than 550 grams.

[0032] The term "assisted labor" refers to a delivery aided by the administration of oxytocin or the artificial rupture of the amniotic sac.

[0033] Clinical-grade cells are defined as cells that have not come into contact with any substance of non-human animal origin, such as non-human animal serum, during cell isolation, culture, cryopreservation, and thawing. Clinical-grade cells are produced under culture conditions compatible with human use and compliant with Good Pharmaceutical Manufacturing Practices (GMP).

[0034] The term “cryopreserved stem cells” refers to stem cells preserved at very low temperatures, preferably between -150°C and -196°C, preferably in the presence of a cryoprotective solution containing dimethyl sulfoxide (DMSO).

[0035] Mesenchymal stem cells are multipotent stem cells of mesodermal origin found in various tissues of the adult body, such as bone marrow, adipose tissue, and the umbilical cord. These stromal cells are capable of self-renewal and differentiation into cells of the osteoblastic, chondrocyte, and adipocyte lineages, including, but not limited to, cells of bone, cartilage, adipose tissue and bone marrow stroma, smooth muscle, ligaments, and tendons.

[0036] The term “thawed human mesenchymal stem cells” refers to mesenchymal stem cells isolated from human tissues and having undergone the thawing process of cryopreserved stem cells.

[0037] The term "fresh human mesenchymal stem cells" refers to mesenchymal stem cells isolated from human tissues and which have not undergone any freeze-thaw process.

[0038] Wharton's jelly refers to the extra-embryonic mesoblastic connective tissue that surrounds the two umbilical arteries and vein, thus protecting the umbilical cord. Sepsis is a serious systemic infection of the body caused by pathogenic germs, particularly bacteria. Sepsis is defined as organ dysfunction secondary to an inappropriate bodily response to an infection. Depending on its severity, the septic state can be classified into two categories: sepsis stricto sensu, and septic shock ("The Third International Consensus Definitions for Sepsis and Septic Shock (Sepsis-3)," JAMA. 2016;315(8):801-810). Sepsis, in its narrowest definition, clinically associates an infection with a SOFA (Sequential Organ Failure Assessment Score) > 2 or an increase in the SOFA score greater than or equal to 2 points if there was organ dysfunction prior to the infection.Septic shock, on the other hand, combines sepsis with the need to use vasopressors until MAP [mean arterial pressure] > 65 mmHg and / or lactate > 2 mmol / L (i.e., 18 mg / dL), despite adequate vascular filling.

[0039] DETAILED DESCRIPTION The present invention relates to mesenchymal stem cells (MSCs) for their use in the treatment of sepsis, in particular septic shock.

[0040] In one embodiment, the MSCs are animal stem cells, preferably mammalian, preferably human. In a particular embodiment, the MSCs are human stem cells. In one embodiment, the MSCs are derived from Wharton's jelly, bone marrow, and / or adipose tissue. In one embodiment, the MSCs are derived from Wharton's jelly. In one embodiment, the MSCs are derived from bone marrow. In one embodiment, the MSCs are derived from adipose tissue. Preferably, the MSCs are derived from Wharton's jelly.

[0041] In one embodiment, the MSCs are thawed; they have undergone a freeze-thaw process. More specifically, the present invention relates to thawed human mesenchymal stem cells derived from Wharton's jelly (GW-MSCs) for use in the treatment of sepsis, particularly septic shock.

[0042] In one embodiment, thawed MSCs, preferably thawed MSC-GWs, exhibit phenotypic characteristics different from fresh MSCs, preferably fresh MSC-GWs.

[0043] In one embodiment, thawed CSMs, preferably thawed CSM-GWs, are characterized in that:

[0044] - at least 60% of the cells express the following antigens: CD90, CD73, CD105, CD44; and

[0045] - at least 80% of the cells do not express any of the following markers: CD34, CD1lb, CD19, CD45, HLA-DR; and

[0046] - at least 10% of the cells express the CD106 marker. In one embodiment, thawed MSCs, preferably thawed GW-MSCs, are characterized in that:

[0047] - at least 60% of the cells express the following antigens: CD90, CD73, CD105, CD44; and

[0048] - at least 80% of the cells do not express any of the following markers: CD34, CD1b, CD19, CD45, CD144, HLA-DR; and

[0049] - at least 10% of the cells express the CD106 marker.

[0050] In one embodiment, thawed MSCs, preferably thawed GW-MS, are characterized in that the expression level of at least one marker selected from CD90, CD73, CD105, CD29, CD44, CD146, CD166, HLA-ABC is at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% or more lower than the expression level of the same marker in fresh MSCs, preferably in fresh GW-MS. In one embodiment, thawed MSCs, preferably thawed GW-MS, are characterized in that the expression level of the CD90 marker is at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% or more lower than the expression level of the CD90 marker in fresh MSCs, preferably in fresh GW-MS.

[0051] The term “CD90” refers to the membrane glycoprotein Thy-1, an example of which is the human CD90 protein with UniProtKB accession number P04216.

[0052] In one embodiment, thawed MSCs, preferably thawed GW-MSCs, are characterized in that the expression level of the CD73 marker is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% or more lower than the expression level of the CD73 marker in fresh MSCs, preferably fresh GW-MSCs. "CD73" refers to the ecto-S'-nucleotidase (or NT5E) enzyme, an example of which is the human CD73 protein with UniProtKB accession number P21589.

[0053] In one embodiment, thawed MSCs, preferably thawed GW-MS, are characterized in that the expression level of the CD 105 marker is at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% or more lower than the expression level of the CD 105 marker in fresh MSCs, preferably in fresh GW-MS.

[0054] The term "CD105" refers to the membrane glycoprotein endoglin, an example of which is the human CD105 protein, UniProtKB accession number PI 7813. In one embodiment, thawed MSCs, preferably thawed GW-MS, are characterized in that the expression level of the CD29 marker is at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% or more lower than the expression level of the CD29 marker in fresh MSCs, preferably in fresh GW-MS.

[0055] The term "CD29" refers to the integrin β-l protein, an example of which is the human CD29 protein with UniProtKB accession number P0S556. In one embodiment, thawed MSCs, preferably thawed GW-MS, are characterized in that the expression level of the CD44 marker is at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% or more lower than the expression level of the CD44 marker in fresh MSCs, preferably in fresh GW-MS.

[0056] The term “CD44” refers to the hyaluronan receptor, an example of which is the human CD44 protein with UniProtKB accession number P16070.

[0057] In one embodiment, thawed MSCs, preferably thawed GW-MS, are characterized in that the expression level of the CD 146 marker is at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% or more lower than the expression level of the CD 146 marker in fresh MSCs, preferably in fresh GW-MS.

[0058] The term “CD146” refers to the melanoma cell adhesion molecule (MCAM: Melanoma Cell Adhesion Molecule), an example of which is the human CD146 protein with UniProtKB accession number P43121.

[0059] In one embodiment, thawed MSCs, preferably thawed GW-MSCs, are characterized in that the expression level of the CD166 marker is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% lower than the expression level of the CD166 marker in fresh MSCs, preferably fresh GW-MSCs. CD166 is defined as the activated leukocyte cell adhesion molecule (ALCAM), an example of which is the human CD166 protein with UniProtKB accession number Q13740.

[0060] In one embodiment, thawed MSCs, preferably thawed GW-MSCs, are characterized in that the expression level of the HLA-ABC marker is at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% or more lower than the expression level of the HLA-ABC marker in fresh MSCs, preferably in fresh GW-MSCs. "HLA-ABC" refers to the surface receptors of major histocompatibility complex class I, an example of which is human HLA-ABC, UniProtKB accession number 019689.

[0061] In one embodiment, thawed MSCs, preferably thawed GW-MS, are characterized in that the expression level of at least one marker selected from CD13 and Sox-2 is at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% or more higher than the expression level of the same marker in fresh MSCs, preferably in fresh GW-MS. In one embodiment, thawed MSCs, preferably thawed GW-MS, are characterized in that the expression level of the CD13 marker is at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% or more higher than the expression level of the CD13 marker in fresh MSCs, preferably in fresh GW-MS.

[0062] The term "CD13" refers to membrane alanyl aminopeptidase, an example of which is the human CD13 protein with UniProtKB accession number P15144. In one embodiment, thawed MSCs, preferably thawed GW-MS, are characterized in that the expression level of the Sox-2 marker is at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% or more higher than the expression level of the Sox-2 marker in fresh MSCs, preferably in fresh GW-MS.

[0063] The term "Sox-2" refers to the SRY-box 2 transcription factor, an example of which is the human Sox-2 protein with UniProtKB accession number P48431.

[0064] In one embodiment, thawed MSCs, preferably thawed GW-MS, after at least one subculture, are characterized in that the expression level of at least one marker selected from CD44 and SSEA-4 is at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% or more lower than the expression level of the same marker in fresh MSCs, preferably in fresh GW-MS.

[0065] In one embodiment, thawed MSCs, preferably thawed GW-MS, after at least one subculture, are characterized in that the CD44 marker expression level is at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% or more lower than the CD44 marker expression level in fresh MSCs, preferably in fresh GW-MS.

[0066] In one embodiment, thawed MSCs, preferably thawed GW-MSCs, after at least one subculture, are characterized in that the expression level of the SSEA-4 marker is at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% or more lower than the expression level of the SSEA-4 marker in fresh MSCs, preferably fresh GW-MSCs. "SSEA-4" is defined as the membrane ganglioside consisting of a glycosphingolipid comprising a terminal sialic acid residue (in English, Stage - Specifies Embryonic Antigen 4).

[0067] In one embodiment, thawed MSCs, preferably thawed GW-SCs, after at least one subculture, are characterized in that the expression level of at least one marker selected from CD90, CD166 and HLA-ABC is at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% or more higher than the expression level of the same marker in fresh MSCs, preferably in fresh GW-SCs.

[0068] In one embodiment, thawed MSCs, preferably thawed GW-MS, after at least one subculture, are characterized in that the expression level of the CD90 marker is at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% or more higher than the expression level of the CD90 marker in fresh MSCs, preferably in fresh GW-MS.

[0069] In one embodiment, thawed MSCs, preferably thawed GW-MS, after at least one subculture, are characterized in that the expression level of the CD 166 marker is at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% or more higher than the expression level of the CD 166 marker in fresh MSCs, preferably in fresh GW-MS.

[0070] In one embodiment, thawed MSCs, preferably thawed GW-MSCs, after at least one subculture, are characterized in that the expression level of the HLA-ABC marker is at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% or more higher than the expression level of the HLA-ABC marker in fresh MSCs, preferably fresh GW-MSCs. In one embodiment, the expression level of the markers is measured by methods well known to those skilled in the art. In one embodiment, the expression level of the markers is measured by flow cytometry. In one embodiment, the expression level of the markers is measured by mean fluorescence intensity (MFI).

[0071] In one embodiment, thawed MSCs, preferably thawed GW-MSCs, are characterized by differential protein expression compared to fresh MSCs, preferably fresh GW-MSCs. In one embodiment, thawed MSCs, preferably thawed GW-MSCs, express at least one protein selected from the group comprising or consisting of ACTB (cytoplasmic actin 1), ANXA1 (annexin Al), CAPZB (F-actin-capping protein subunit beta), LASP1 (LIM and SH3 domain protein 1), PRDX2 (peroxiredoxin-2), PRDX3 (mitochondrial thioredoxin-dependent peroxide reductase), PSA3 (proteasome subunit alpha type-3), RS12 (40S ribosomal protein S12), and SYWC (cytoplasmic tryptophan-tRNA ligase).

[0072] In one embodiment, fresh MSCs, preferably fresh MSC-GW, do not express or substantially do not express at least one protein selected from the group comprising or consisting of ACTB (cytoplasmic actin 1), ANXA1 (annexin Al), CAPZB (F-actin-capping protein subunit beta), LASP1 (LIM and SH3 domain protein 1), PRDX2 (peroxiredoxin-2), PRDX3 (mitochondrial thioredoxin-dependent peroxide reductase), PSA3 (proteasome subunit alpha type-3), RS12 (40S ribosomal protein S 12) and SYWC (cytoplasmic tryptophan-tRNA ligase).

[0073] In one embodiment, thawed MSCs, preferably thawed GW-MS, express at least 1.1 times, 1.2 times, 1.3 times, 1.4 times, 1.5 times, 1.6 times, 1.7 times, 1.8 times, 1.9 times, 2 times, 2.5 times, 3.0 times, 3.5 times, 4.0 times, 4.5 times, 5.0 times, 7.5 times, 10.0 times, 15.0 times, 20.0 times, 30.0 times, 40.0 times, 50.0 times more or less one protein selected from the group comprising or consisting of ACTB (cytoplasmic actin 1), ANXA1 (annexin Al), CAPZB (F-actin-capping protein subunit beta), LASP1 (LIM and SH3 domain protein 1), PRDX2 (peroxiredoxin-2), PRDX3 (mitochondrial thioredoxin-dependent peroxide reductase), PSA3 (proteasome subunit alpha type-3), RS12 (40S ribosomal protein S 12) and SYWC (cytoplasmic tryptophan-tRNA ligase), than fresh MSCs, preferably than fresh MSC-GW.The term "ACTB" refers to cytoplasmic actin 1 protein, an example of which is human ACTB with UniProtKB accession number P60709.

[0074] "ANXA1" refers to annexin 1 protein (in English, annexin Al), an example of which is human ANXA1 with UniProtKB accession number P04083. "CAPZB" refers to the β subunit of the Pacin F capping protein (in English, F-actin-capping protein subunit beta), an example of which is human CAPZB with UniProtKB accession number P47756.

[0075] The term "LASP1" refers to LIM and SH3 domain protein 1, an example of which is human LASP1 with UniProtKB accession number Q14847.

[0076] The term "PRDX2" refers to peroxiredoxin 2 (in English, peroxiredoxin-2). ' ), one example of which is human PRDX2, whose UniProtKB accession number is P32119.

[0077] The term “PRDX3” refers to peroxiredoxin 3 (in English, mitochondrial thioredoxin- dependent peroxide reductase), an example of which is human PRDX3 with UniProtKB accession number P30048.

[0078] The term “PSA3” refers to the type 3 proteasome subunit (in English, proteasome subunit alpha type-3), an example of which is human PS A3, whose UniProtKB accession number is P25788.

[0079] RS12 refers to the 40S ribosomal protein S12, an example of which is human RS12 with UniProtKB accession number P25398. SYWC refers to cytoplasmic tryptophan-tRNA synthetase, an example of which is human SYWC with UniProtKB accession number P23381.

[0080] In one embodiment, thawed MSCs, preferably thawed GW-SCs, do not express or substantially do not express at least one protein selected from the group comprising or consisting of ACTS (skeletal muscle alpha actin), ALIBI (mitochondrial aldehyde dehydrogenase X), ANX10 (annexin A10), GBB1 (guanine nucleotide-binding protein G(I) / G(S) / G(T) subunit beta-1), GBB2 (guanine nucleotide-binding protein G(I) / G(S) / G(T) subunit beta-2), GPRIN1 (G protein-regulated inducer of neurite outgrowth 1), DTNA (dystrobrevin alpha), MIPOl (mirror-image polydactyly gene 1 protein), PSB3 (proteasome subunit beta type-3) and PSDE (26S proteasome non-ATPase regulatory subunit 14).

[0081] In one embodiment, fresh MSCs, preferably fresh MSC-GWs, express at least one protein selected from the group comprising or consisting of ACTS (skeletal muscle alpha actin), ALIBI (mitochondrial aldehyde dehydrogenase X), ANX10 (annexin A10), GBB1 (guanine nucleotide-binding protein G(I) / G(S) / G(T) subunit beta-1), GBB2 (guanine nucleotide-binding protein G(I) / G(S) / G(T) subunit beta-2), GPRIN1 (G protein-regulated inducer of neurite outgrowth 1), DTNA (dystrobrevin alpha), MIPOl (mirror-image polydactyly gene 1 protein), PSB3 (proteasome subunit beta type-3) and PSDE (26S proteasome non-ATPase regulatory subunit 14).

[0082] In one embodiment, thawed MSCs, preferably thawed GW-MSCs, express at least 1.1 times, 1.2 times, 1.3 times, 1.4 times, 1.5 times, 1.6 times, 1.7 times, 1.8 times, 1.9 times, 2 times, 2.5 times, 3.0 times, 3.5 times, 4.0 times, 4.5 times, 5.0 times, 7.5 times, 10.0 times, 15.0 times, 20.0 times, 30.0 times, 40.0 times, or 50.0 times less at least one protein selected from the group comprising or consisting of ACTS (skeletal muscle alpha actin), ALIBI (mitochondrial aldehyde dehydrogenase X), ANX10 (annexin A10), or GBB1 (guanine nucleotide-binding protein). G(T) / G(S) / G(T) subunit beta- 1), GBB2 (guanine nucleotide-binding protein G(I) / G(S) / G(T) subunit beta-2), GPRIN1 (G protein-regulated inducer of neurite outgrowth 1), DTNA (dystrobrevin alpha), MIPOl (mirror-image polydactyly gene 1 protein), PSB3 (proteasome subunit beta type-3) and PSDE (26S proteasome non-ATPase regulatory subunit 14), than fresh MSCs, preferably than fresh GW-MSCs.

[0083] The term “ACTS” refers to the skeletal muscle alpha actin protein, an example of which is human ACTS with UniProtKB accession number P68133.

[0084] The term "ALIBI" refers to mitochondrial aldehyde dehydrogenase X, an example of which is human ALIBI with UniProtKB accession number P30837. The term "ANX10" refers to annexin A10 protein, an example of which is human ANX10 with UniProtKB accession number Q9UJ72.

[0085] The term "GBB1" refers to the βΐ subunit of the guanine nucleotide-binding protein G(I) / G(S) / G(T) subunit beta-1, an example of which is human GBB1 with UniProtKB accession number P62873.

[0086] "GBB2" refers to the β2 subunit of the guanine nucleotide-binding protein G(I) / G(S) / G(T) subunit beta-2, an example of which is human GBB2 with UniProtKB accession number P62879. "GPRIN1" refers to the G protein-regulated inducer of neurite outgrowth 1, an example of which is human GPRIN1 with UniProtKB accession number Q7Z2K8.

[0087] The term "DTNA" refers to dystrobrevin alpha, an example of which is human DTNA with UniProtKB accession number Q9Y4J8. "MIPOl" refers to the mirror-image polydactyly gene 1 protein, an example of which is human MIPOl with UniProtKB accession number Q8TD10. "PSB3" refers to the proteasome subunit beta type-3, an example of which is human PSB3 with UniProtKB accession number P49720.

[0088] The term “PSDE” refers to the 26S proteasome non-ATPase regulatory subunit 14, an example of which is the human PSDE with UniProtKB accession number 000487.

[0089] In one embodiment, thawed MSCs, preferably thawed GW-MSCs, after at least 1 subculture, express at least 1.1 times, 1.2 times, 1.3 times, 1.4 times, 1.5 times, 1.6 times, 1.7 times, 1.8 times, 1.9 times, 2 times, 2.5 times, 3.0 times, 3.5 times, 4.0 times, 4.5 times, 5.0 times, 7.5 times, 10.0 times, 15.0 times, 20.0 times, 30.0 times, 40.0 times, 50.0 times more or less one protein selected from the group comprising or consisting of AMPM2 (methionine aminopeptidase 2), DJB11 (dnaJ homolog subfamily B member 11), K1C18 (keratin, type I cytoskeletal 18), K1C19 (keratin, type I cytoskeletal 19), K2C8 (keratin type II cytoskeletal 8), LYSC (lysozyme C), PDIA3 (protein disulfide-isomerase A3), TCTP (translationally-controlled tumor protein) and TGM2 (protein-glutamine gamma-glutamyltransferase 2) than fresh MSCs, preferably than fresh MSC-GW.

[0090] The term “AMPM2” refers to methionine aminopeptidase 2, an example of which is human AMPM2 with UniProtKB accession number P50579.

[0091] "DJB11" refers to member 11 of the HSP40 homolog subfamily B (dnaJ homolog subfamily B member 11), an example of which is human DJB11 with UniProtKB accession number Q9UBS4. "K1C18" refers to acidic type I cytokeratin 18 (keratin, type I cytoskeletal 18), an example of which is human K1C18 with UniProtKB accession number P05783. "K1C19" refers to acidic type I cytokeratin 19 (keratin, type I cytoskeletal 19), an example of which is human K1C19 with UniProtKB accession number P08727.

[0092] "K2C8" refers to basic type II cytokeratin 8 (in English, keratin type II cytoskeletal 8), an example of which is human K2C8 with UniProtKB accession number P05787.

[0093] The term "LYSC" refers to lysozyme C, an example of which is human LYSC, whose UniProtKB accession number is P61626.

[0094] The term “PDIA3” refers to the protein disulfide isomerase A3 enzyme, an example of which is human PDIA3 with UniProtKB accession number P30101.

[0095] The term "TCTP" refers to translationally-controlled tumor protein, an example of which is human TCTP with UniProtKB accession number P13693. The term "TGM2" refers to the enzyme γ-glutamyltranspeptidase 2 (protein-glutamine gamma-glutamyltransferase 2), an example of which is human TGM2 with UniProtKB accession number P21980.

[0096] In one embodiment, thawed MSCs, preferably thawed GW-MSCs, after at least one subculture, express at least 1.1 times, 1.2 times, 1.3 times, 1.4 times, 1.5 times, 1.6 times, 1.7 times, 1.8 times, 1.9 times, 2 times, 2.5 times, 3.0 times, 3.5 times, 4.0 times, 4.5 times, 5.0 times, 7.5 times, 10.0 times, 15.0 times, 20.0 times, 30.0 times, 40.0 times, or 50.0 times less at least one protein selected from the group comprising or consisting of GSTP1 (glutathione S-transferase P), HSP7C (heat shock cognate 71 kDa protein), HSPB1 (heat shock protein beta-1), or LEG1. (galectin-1), S10AB (protein S100-A11) and UBE2N (ubiquitin-conjugating enzyme E2 N), rather than fresh MSCs, preferably fresh GW-MSCs. "GSTP1" refers to the enzyme glutathione S-transferase P, an example of which is human GSTP1 with UniProtKB accession number P09211.

[0097] The term "HSP7C" refers to heat shock cognate 71 kDa protein, an example of which is human HSP7C with UniProtKB accession number PI 1142.

[0098] "HSPB1" refers to heat shock protein 27 (in English, heat shock protein beta-J), an example of which is human HSPB1 with UniProtKB accession number P04792. "LEG1" refers to galectin 1 (in English, galectin-1), an example of which is human LEG1 with UniProtKB accession number P09382.

[0099] The term "S10AB" refers to the S100-A11 protein (in English, protein SJ00-A11), an example of which is human S10AB, whose UniProtKB accession number is P31949.

[0100] "UBE2N" refers to the ubiquitin-conjugating enzyme E2 N, an example of which is human UBE2N with UniProtKB accession number P61088.

[0101] In one embodiment, protein expression is measured by methods well known to those skilled in the art. In another embodiment, protein expression is measured by mass spectrometry.

[0102] In one embodiment, thawed MSCs, preferably thawed GW-SCs, under in vitro and / or non-inflammatory conditions, secrete at least one growth factor selected from BMP-7, IGFBP-1, insulin, FGF-7, NT-4 and VEGF-D.

[0103] In one embodiment, thawed MSCs, preferably thawed GW-MSCs, under in vitro and / or non-inflammatory conditions, secrete at least 1.1 times, 1.2 times, 1.3 times, 1.4 times, 1.5 times, 1.6 times, 1.7 times, 1.8 times, 1.9 times, 2 times, 2.5 times, 3.0 times, 3.5 times, 4.0 times, 4.5 times, 5.0 times, 7.5 times, 10.0 times, 15.0 times, 20.0 times, 30.0 times, 40.0 times, or 50.0 times more at least one growth factor selected from BMP-7, IGFBP-1, insulin, FGF-7, NT-4, and VEGF-D than fresh MSCs, preferably fresh GW-MSCs, under in vitro conditions. and / or in a non-inflammatory condition.

[0104] In one embodiment, thawed MSCs, preferably thawed GW-MSCs, under in vitro and / or non-inflammatory conditions, secrete at least 50 pg / mL, at least 100 pg / mL, at least 150 pg / mL, at least 200 pg / mL, at least 250 pg / mL, at least 300 pg / mL, at least 350 pg / mL, at least 400 pg / mL, at least 450 pg / mL, at least 500 pg / mL or more of BMP-7.

[0105] In one embodiment, thawed MSCs, preferably thawed GW-MSCs, under in vitro and / or non-inflammatory conditions, secrete at least 20 pg / mL, at least 25 pg / mL, at least 30 pg / mL, at least 35 pg / mL, at least 40 pg / mL, at least 45 pg / mL, at least 50 pg / mL, at least 55 pg / mL, at least 60 pg / mL, at least 65 pg / mL or more of IGFBP-1.

[0106] In one embodiment, thawed MSCs, preferably thawed GW-MSCs, under in vitro and / or non-inflammatory conditions, secrete at least 50 pg / mL, at least 75 pg / mL, at least 100 pg / mL, at least 125 pg / mL, at least 150 pg / mL, at least 175 pg / mL, at least 200 pg / mL, at least 225 pg / mL, at least 250 pg / mL, at least 275 pg / mL or more of insulin.

[0107] In one embodiment, thawed MSCs, preferably thawed GW-MSCs, under in vitro and / or non-inflammatory conditions, secrete at least 10 pg / mL, at least 15 pg / mL, at least 20 pg / mL, at least 25 pg / mL, at least 30 pg / mL, at least 35 pg / mL, at least 40 pg / mL, at least 45 pg / mL, at least 50 pg / mL, at least 55 pg / mL, at least 60 pg / mL, at least 65 pg / mL or more of FGF-7.

[0108] In one embodiment, thawed MSCs, preferably thawed GW-MSCs, under in vitro and / or non-inflammatory conditions, secrete at least 45 pg / mL, at least 50 pg / mL, at least 55 pg / mL, at least 60 pg / mL, at least 65 pg / mL, at least 70 pg / mL, at least 75 pg / mL, at least 80 pg / mL, at least 85 pg / mL, at least 90 pg / mL or more of NT-4.

[0109] In one embodiment, thawed MSCs, preferably thawed GW-MSCs, under in vitro and / or non-inflammatory conditions, secrete at least 5 pg / mL, at least 10 pg / mL, at least 15 pg / mL, at least 20 pg / mL, at least 25 pg / mL, at least 30 pg / mL, at least 35 pg / mL, at least 40 pg / mL, at least 45 pg / mL, at least 50 pg / mL or more of VEGF-D.

[0110] In one embodiment, thawed MSCs, preferably thawed GW-MSCs, under in vivo and / or inflammatory conditions, secrete at least one growth factor selected from BMP-7 and TGFP3.

[0111] In one embodiment, thawed MSCs, preferably thawed GW-MS, under in vivo and / or inflammatory conditions, secrete at least 1.1 times, 1.2 times, 1.3 times, 1.4 times, 1.5 times, 1.6 times, 1.7 times, 1.8 times, 1.9 times, 2 times, 2.5 times, 3.0 times, 3.5 times, 4.0 times, 4.5 times, 5.0 times, 7.5 times, 10.0 times, 15.0 times, 20.0 times, 30.0 times, 40.0 times, 50.0 times more of at least one growth factor selected from BMP-7 and TGFP3, than fresh MSCs, preferably fresh GW-MS, under in vivo and / or inflammatory conditions. In one embodiment, thawed MSCs, preferably thawed GW-MSCs, under in vivo and / or inflammatory conditions, secrete at least 200 pg / mL, at least 250 pg / mL, at least 300 pg / mL, at least 350 pg / mL, at least 400 pg / mL, at least 450 pg / mL, at least 500 pg / mL, at least 550 pg / mL, at least 600 pg / mL, at least 650 pg / mL, at least 700 pg / mL, at least 750 pg / mL or more of BMP-7.

[0112] In one embodiment, thawed MSCs, preferably thawed GW-MSCs, under in vivo and / or inflammatory conditions, secrete at least 20 pg / mL, at least 25 pg / mL, at least 30 pg / mL, at least 35 pg / mL, at least 40 pg / mL, at least 45 pg / mL, at least 50 pg / mL, at least 55 pg / mL, at least 60 pg / mL, at least 65 pg / mL or more TGFp3.

[0113] In one embodiment, thawed MSCs, preferably thawed GW-SCs, under in vivo and / or inflammatory conditions, do not secrete or substantially do not secrete IGFBP-1.

[0114] In one embodiment, thawed MSCs, preferably thawed GW-MSCs, under in vivo and / or inflammatory conditions, secrete at least 1.1 times, 1.2 times, 1.3 times, 1.4 times, 1.5 times, 1.6 times, 1.7 times, 1.8 times, 1.9 times, 2 times, 2.5 times, 3.0 times, 3.5 times, 4.0 times, 4.5 times, 5.0 times, 7.5 times, 10.0 times, 15.0 times, 20.0 times, 30.0 times, 40.0 times, 50.0 times less IGFBP-1 than fresh MSCs, preferably fresh GW-MSCs, under in vivo and / or inflammatory conditions.

[0115] In one embodiment, thawed MSCs, preferably thawed GW-MSCs, under in vivo and / or inflammatory conditions, secrete less than 20 pg / mL, at least 15 pg / mL, at least 10 pg / mL, at least 5 pg / mL, at least 4 pg / mL, at least 3 pg / mL, at least 2 pg / mL, at least 1 pg / mL or less IGFBP-1.

[0116] The term "BMP-7" refers to bone morphogenetic protein 7. An example of BMP-7 is the human BMP-7 protein, whose UniProtKB accession number is P 18075.

[0117] IGFBP-1 refers to insulin-like growth factor-binding protein 1. An example of IGFBP-1 is the human IGFBP-1 protein with UniProtKB accession number P08833. Insulin refers to the protein hormone that facilitates the absorption of blood glucose. An example of insulin is human insulin with UniProtKB accession number P01308. FGF-7 refers to fibroblast growth factor 7. An example of FGF-7 is the human FGF-7 protein with UniProtKB accession number P21781.

[0118] The term "NT-4" refers to neurotrophin 4. An example of NT-4 is the human NT-4 protein, whose UniProtKB accession number is P34130.

[0119] VEGF-D refers to vascular endothelial growth factor D. An example of VEGF-D is the human VEGF-D protein, UniProtKB accession number 043915.

[0120] The term "TGFP3" refers to Transforming Growth Factor β3. An example of TGFP3 is the human TGFP3 protein, whose UniProtKB accession number is PI 0600.

[0121] The term "in vitro condition" refers to a condition in which thawed MSCs, preferably thawed GW-MSCs, are outside a living organism, preferably outside the patient. The term "in vivo condition" refers to a condition in which thawed MSCs, preferably thawed GW-MSCs, are inside a living organism, preferably inside the patient, after their administration to the patient.

[0122] A "non-inflammatory condition" is defined as a condition in which thawed MSCs, preferably thawed GW-MSCs, are not stimulated, in vitro or in vivo, by inflammatory cytokines, including but not limited to TNF-α and IFN-γ. Specifically, a non-inflammatory condition can be defined as an in vivo condition, preferably within the patient, said patient being healthy or substantially healthy, and said patient not affected by sepsis.

[0123] An "inflammatory condition" is defined as a condition in which thawed MSCs, preferably thawed GW-MSCs, are stimulated, in vitro or in vivo, by inflammatory cytokines, including but not limited to TNF-α and IFN-γ. Specifically, an inflammatory condition can be defined as an in vivo condition, preferably within the patient, said patient being affected by sepsis. In one embodiment, the thawed MSCs, preferably thawed GW-MSCs, secrete VEGF.

[0124] In one embodiment, thawed MSCs, preferably thawed MSC-GWs, secrete at least 1.1 times, 1.2 times, 1.3 times, 1.4 times, 1.5 times, 1.6 times, 1.7 times, 1.8 times, 1.9 times, 2 times, 2.5 times, 3.0 times, 3.5 times, 4.0 times, 4.5 times, 5.0 times, 7.5 times, 10.0 times, 15.0 times, 20.0 times, 30.0 times, 40.0 times, 50.0 times more VEGF than fresh MSCs, preferably fresh MSC-GWs.

[0125] In one embodiment, thawed MSCs, preferably thawed GW-MSCs, secrete at least 500 pg / mL, at least 600 pg / mL, at least 700 pg / mL, at least 800 pg / mL, at least 900 pg / mL, at least 1 ng / mL, at least 1.25 ng / mL, at least 1.5 ng / mL, at least 1.75 ng / mL, at least 2 ng / mL or more of VEGF.

[0126] In one embodiment, thawed MSCs, preferably thawed MSC-GWs, do not secrete or substantially do not secrete VEGF.

[0127] In one embodiment, thawed MSCs, preferably thawed MSC-GWs, secrete at least 1.1 times, 1.2 times, 1.3 times, 1.4 times, 1.5 times, 1.6 times, 1.7 times, 1.8 times, 1.9 times, 2 times, 2.5 times, 3.0 times, 3.5 times, 4.0 times, 4.5 times, 5.0 times, 7.5 times, 10.0 times, 15.0 times, 20.0 times, 30.0 times, 40.0 times, 50.0 times less VEGF than fresh MSCs, preferably fresh MSC-GWs.

[0128] In one embodiment, thawed MSCs, preferably thawed MSC-GWs, secrete less than 500 pg / mL, at least 400 pg / mL, at least 300 pg / mL, at least 250 pg / mL, at least 200 pg / mL, at least 150 pg / mL, at least 100 pg / mL, at least 50 pg / mL or less of VEGF.

[0129] VEGF stands for Vascular Endothelial Growth Factor. An example of VEGF is the human VEGF protein, UniProtKB accession number PI 5692.

[0130] In one embodiment, thawed MSCs, preferably thawed GW-MSCs, under in vivo and / or inflammatory conditions, induce VEGF secretion. Several cell types can secrete VEGF. Examples of such cells include, but are not limited to, macrophages, monocytes, endothelial cells, myofibroblasts, chondrocytes, and hematopoietic cells.

[0131] In one embodiment, thawed MSCs, preferably thawed MSC-GWs, induce an increase in serum VEGF concentration of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or more, compared to fresh MSCs, preferably compared to fresh MSC-GWs.

[0132] In one embodiment, thawed MSCs, preferably thawed MSC-GWs, induce an increase in plasma VEGF concentration of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or more, compared to fresh MSCs, preferably compared to fresh MSC-GWs.

[0133] In one embodiment, the MSCs, preferably GW-MSCs, are stored at very low temperatures. In another embodiment, the MSCs, preferably GW-MSCs, are stored at approximately -20°C, preferably at approximately -80°C, and preferably between -150°C and -196°C. In another embodiment, the MSCs, preferably GW-MSCs, are stored in the presence of a cryoprotectant solution. Cryoprotectant cell solutions are well known to those skilled in the art. Such solutions include, e.g., in one embodiment, the cryoprotectant solution comprises at least one cryoprotectant selected from dimethyl sulfoxide (DMSO), glycerol, ethylene glycol, propylene glycol, formamide, butene diol, and mixtures thereof.

[0134] In one embodiment, the MSCs, preferably MSC-GWs, are thawed according to a conventional thawing protocol. For example, the MSCs, preferably MSC-GWs, are thawed in a water bath at 37°C and then washed in the presence of a washing solution comprising NaCl, ralbumin and dextrose citrate formula A (ACD).

[0135] A particular embodiment of the invention relates to thawed CSM-GW as described above, for their use in the treatment of septic shock, the most serious form of sepsis, which is defined by the appearance or persistence of arterial hypotension and / or signs of peripheral hypoperfusion despite adequate vascular filling.

[0136] In an advantageous embodiment, the MSCs, preferably the MSC-GWs used in the invention, are directly obtained from thawing, without re-culturing after thawing.

[0137] Unlike previous studies, in which the cells analyzed were fresh cells, which had not been subjected to freezing or thawing, and administered at early times to animals in a septic shock model, the results of the present invention provide for the first time evidence of the efficacy of GW-MSCs under conditions compatible with clinical conditions, namely the administration of thawed GW-MSCs at a late time in the evolution of sepsis.

[0138] The present invention shows that thawed GSM-GW retain immunomodulatory and antibacterial properties at a level comparable to those of thawed bone marrow-isolated MSCs (BM-SMs) and that administration of GSM-GW even increases the survival of mice suffering from septic shock compared to the survival of mice treated with BM-SMs.

[0139] These results show that thawed CSM-GW are suitable for immediate use under clinical conditions for the treatment of septic shock.

[0140] In one embodiment, the CSMs, preferably the CSM-GWs used in the invention, are washed after thawing. In another embodiment, the CSMs, preferably the CSM-GWs used in the invention, are resuspended after thawing. In another embodiment, the CSMs, preferably the CSM-GWs used in the invention, are resuspended after thawing in a hydroxyethyl starch solution. In another embodiment, the CSMs, preferably the CSM-GWs used in the invention, are resuspended after thawing in an albumin solution. In one embodiment, the albumin solution is a 4% albumin solution. In another embodiment, the albumin solution further comprises NaCl and / or dextrose citrate formula A (ACD).

[0141] In the present invention, the Inventors also note for the first time a significant correlation between, on the one hand, obstetric factors and, on the other hand, the proliferation of CSM-GW and make available the criteria for the choice of umbilical cords to obtain CSM-GW with a better capacity for proliferation.

[0142] One of the objects of the invention relates to GW-MSCs derived from human umbilical cord tissue meeting these criteria. It is observed that these criteria improve cell proliferation by decreasing the cell doubling time, particularly at the Pl transition. These criteria also allow for obtaining a greater number of cells with a short doubling time.

[0143] In an advantageous embodiment, the GW-MSCs used in the present invention are derived from human umbilical cord tissue from a mother meeting at least one of the following criteria: having received oxytocin during delivery, having delivered by assisted labor, having delivered at term, not having experienced preeclampsia during pregnancy, whose child did not present with neonatal disorders, and having been exposed to tobacco smoke during pregnancy. The inventors have found that these obstetric factors have a positive impact on the cell proliferation of MSCs, preferably GW-MSCs, and allow for the selection of MSCs, preferably GW-MSCs, with improved proliferation properties.

[0144] According to one embodiment, the MSCs, preferably the GW-MSCs used in the present invention, are clinical grade cells.

[0145] Therefore, all culture media and reagents used in the present invention are free of substances of non-human animal origin. In one embodiment, all culture media and reagents used are free of serum of non-human animal origin. In another embodiment, all culture media and reagents used are free of serum of any origin, even human.

[0146] Advantageously, the culture medium used in the present invention for cell adhesion contains human platelet lysate.

[0147] The present invention also relates to a composition comprising thawed MSCs, preferably thawed Wharton's jelly MSCs as described above, for use in the treatment of sepsis. The present invention further relates to a pharmaceutical composition comprising, as active ingredients, thawed clinical-grade mesenchymal stem cells derived from Wharton's jelly as described above, for use in the treatment of sepsis.

[0148] The composition further comprises a pharmaceutically acceptable excipient. According to the present invention, a pharmaceutically acceptable excipient is an excipient free from substances of non-human animal origin and suitable for use in contact with human cells without toxicity, irritation, or undue allergic response. A person skilled in the art will be able to select a pharmaceutically acceptable excipient according to the pharmaceutical formulation of the composition and its method of administration.

[0149] For example, said excipient is 4% albumin or a 130 / 0.42 hydroxyethyl starch (HES) solution.

[0150] The pharmaceutical composition may be in the form of an infusion product and packaged in an infusion bag. The present invention also relates to a medicinal product comprising thawed MSCs, preferably thawed GW-MSCs as described above, for use in the treatment of sepsis.

[0151] In one embodiment, the MSCs, preferably the MSC-GWs used in the invention, can be administered to the patient systemically or locally.

[0152] In one embodiment, the MSCs, preferably the MSC-GWs used in the invention, can be administered to the patient intravenously, intravascularly, intracerebrically, parenterally, intraperitoneally, epidurally, intraspinally, intrastemally, intraarticularly, intrasynovially, intrathecally, intraarterially, intracardiacly, or intramuscularly.

[0153] In one embodiment, the CSMs, preferably the CSM-GWs used in the invention, can be administered to the patient by bolus injection (also called rapid injection) or by continuous infusion (also called slow injection).

[0154] In a preferred embodiment, thawed clinical-grade CSM-GW for use in the treatment of sepsis are administered intravenously.

[0155] In accordance with the present invention, the previously described thawed clinical-grade CSM-GW can be administered to a patient suffering from sepsis with a dose of approximately 0.3 xlO 6 at 3xl0 6 cells per kg of body weight, including a dose of lxlO 6 cells per kg of body weight. A person skilled in the art can adjust the dose according to the severity of the infection, the patient's weight, and the number or frequency of administration.

[0156] In one embodiment, the CSMs, preferably the CSM-GWs used in the invention, can be administered to the patient at a dose ranging from 10 3 at 10 9 CSM / kg, preferably 10 4 at 10 8 CSM / kg, preferably 10 5 at 10 7 CSM / kg. In one embodiment, the CSMs, preferably the CSM-GW used in the invention, can be administered to the patient at a dose of 1 x 10 s CSM / kg, 2 x 10 5 CSM / kg, 3 x 10 5 CSM / kg, 4 x 10 5 CSM / kg, 5 x 10 5 CSM / kg, 6 x 10 5 CSM / kg, 7 x 10 5 CSM / kg, 8 x 10 5 CSM / kg, 9 x 10 5 CSM / kg, 1 x 10 6 CSM / kg, 2 x 10 6 CSM / kg, 3 x 10 6 CSM / kg, 4 x 10 6 CSM / kg, 5 x 10 6 CSM / kg, 6 x 10 6 CSM / kg, 7 x 10 6 CSM / kg, 8 x 10 6 CSM / kg, 9 x 10 6 CSM / kg, 1 x 10 7 CSM / kg.

[0157] In one embodiment, the CSMs, preferably the CSM-GWs used in the invention, can be administered to the patient at a dose ranging from 10 3 at 10 9 CSM / kg / day, preferably 10 4 at 10 8 CSM / kg / day, preferably 10 5 at 10 7 CSM / kg / day.

[0158] In one embodiment, the MSCs, preferably the MSC-GWs used in the invention, can be administered to the patient at a dose of 1 x 10 5 CSM / kg / day, 2 x

[0159] 10 5 CSM / kg / day, 3 x 10 5 CSM / kg / day, 4 x 10 5 CSM / kg / day, 5 x 10 5 CSM / kg / day, 6 x 10 5 CSM / kg / day, 7 x 10 5 CSM / kg / day, 8 x 10 5 CSM / kg / day, 9 x 10 5 CSM / kg / day, 1 x

[0160] 10 6 CSM / kg / day, 2 x 10 6 CSM / kg / day, 3 x 10 6 CSM / kg / day, 4 x 10 6 CSM / kg / day, 5 x 10 6 CSM / kg / day, 6 x 10 6CSM / kg / day, 7 x 10 6 CSM / kg / day, 8 x 10 6 CSM / kg / day, 9 x 10 6 CSM / kg / day, 1 x 10 7 CSM / kg / day.

[0161] In one embodiment, the CSMs, preferably the CSM-GWs used in the invention, can be administered to the patient at a dose ranging from 10 3 at 10 9 CSM / day, preferably 10 4 at 10 8 CSM / day, preferably 10 5 at 10 7 CSM / day.

[0162] In one embodiment, the MSCs, preferably the MSC-GWs used in the invention, can be administered to the patient at a dose of 1 x 10 s CSM / kg / day, 2 x

[0163] 10 5 CSM / day, 3 x 10 5 CSM / day, 4 x 10 5 CSM / day, 5 x 10 5 CSM / day, 6 x 10 5 CSM / day, 7 x 10 5 CSM / day, 8 x 10 5 CSM / day, 9 x 10 5 CSM / day, 1 x 10 6 CSM / day, 2 x

[0164] 106 CSM / day, 3 x 10 6 CSM / day, 4 x 10 6 CSM / day, 5 x 10 6 CSM / day, 6 x 10 6 CSM / day, 7 x 10 6 CSM / day, 8 x 10 6 CSM / day, 9 x 10 6 CSM / day, 1 x 10 7 CSM / day.

[0165] In one embodiment, the CSMs, preferably the CSM-GWs used in the invention, can be administered in a single dose, or in multiple doses spaced out over time.

[0166] In one embodiment, the CSMs, preferably the CSM-GWs used in the invention, can be administered once a day, twice a day, three times a day, or more. In another embodiment, the CSMs, preferably the CSM-GWs used in the invention, can be administered every day, every two days, every three days, every four days, every five days, or every six days.

[0167] In one embodiment, the CSMs, preferably the CSM-GWs used in the invention, can be administered once a week, every week, every 2 weeks, every 3 weeks.

[0168] In one embodiment, the MSCs, preferably the MSC-GWs used in the invention, can be administered once a month, every month, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, or more frequently. According to the invention, the thawed MSC-GWs described above can be diluted before use in a suitable excipient, for example, a 4% albumin solution (4 g / 100 mL) or a 130 / 0.42 hydroxyethylarnidine (HES) solution.

[0169] Thawed CSM-GW for the treatment of sepsis according to the present invention can be used alone, or before, during, or after other sepsis treatment, including antibiotic treatment.

[0170] In one embodiment, treatments for sepsis are well known to those skilled in the art and include, but are not limited to, antibiotic therapy, antifungal therapy, fluid resuscitation, administration of vasopressors, and corticosteroid therapy. In an advantageous embodiment, thawed clinical-grade MSC-GW for use in the treatment of sepsis is administered to patients with sepsis who are receiving antibiotic therapy.

[0171] In one embodiment, the patient is receiving at least one antibiotic. In another embodiment, the patient is receiving at least two antibiotics. In yet another embodiment, the patient is receiving three or more antibiotics. Examples of antibiotics for the treatment of sepsis include, but are not limited to, ampicillin, azithromycin, aztreonam, cefazolin, cefepime, clindamycin, levofloxacin, linezolid, meropenem, metronidazole, piperacillin, tazobactam, tobramycin, and vancomycin. In one embodiment, thawed clinical-grade MSC-GW for use in the treatment of sepsis is administered to patients with sepsis who are receiving antifungal therapy.

[0172] In one embodiment, the patient is being treated with at least one antifungal. In another embodiment, the patient is being treated with at least two antifungals. In another embodiment, the patient is being treated with three or more antifungals.

[0173] Examples of antifungals for the treatment of sepsis include, but are not limited to, amphotericin B deoxycholate, anidulafungin, caspofungin, fluconazole, itraconazole, micafungin, and voriconazole. In one embodiment, thawed clinical-grade MSC-GW for use in the treatment of sepsis is administered to patients with sepsis who are receiving fluid resuscitation.

[0174] Fluid resuscitation corrects hypovolemia, maintains mean arterial pressure above 65 mmHg, and limits clinical signs of hypoperfusion by restoring intravascular volume. Fluid resuscitation is performed using intravenous solutions.

[0175] Examples of solutions for vascular filling in the treatment of sepsis include, but are not limited to, colloids and crystalloids.

[0176] In one embodiment, thawed clinical-grade CSM-GW for use in the treatment of sepsis is administered to patients with sepsis who are receiving vasopressor therapy. In one embodiment, the patient is receiving at least one vasopressor. In another embodiment, the patient is receiving at least two vasopressors. In yet another embodiment, the patient is receiving three or more vasopressors. Examples of vasopressors for the treatment of sepsis include, but are not limited to, catecholamines (including norepinephrine and epinephrine) and vasopressin.

[0177] In one embodiment, thawed clinical-grade CSM-GW for use in the treatment of sepsis is administered to patients with sepsis who are receiving corticosteroid therapy. In one embodiment, the patient is receiving at least one corticosteroid. In another embodiment, the patient is receiving at least two corticosteroids. In another embodiment, the patient is receiving three or more corticosteroids.

[0178] Examples of corticosteroids for the treatment of sepsis include, but are not limited to, cortisone, hydrocortisone, and prednisone. Other treatments are suitable for sepsis and are well known to those skilled in the art. Examples of such treatments include, but are not limited to, PAB103, recombinant alkaline phosphatase recAP, CaCP29, activated drotrecogin alfa, GEA-230, eritoran, esmolol, GM-CSF, IFNγ, lenercept, levosimendan, LR12, selepressin, talactoferrin alpha, and recombinant human thrombomodulin.

[0179] Another aspect of the invention relates to a method for preparing clinical-grade mesenchymal stem cells from Wharton's jelly. The method of the invention aims to produce Wharton's jelly-derived mesenchymal stem cells (MSCs) with improved proliferative capacity.

[0180] In one embodiment, the process of the invention aims to produce CSM-GWs as defined in the present invention. In one embodiment, the process of the invention aims to produce CSM-GWs as defined in the present invention for use in the treatment of sepsis. Said process comprises the following steps:

[0181] (i) to cultivate human umbilical cord tissue containing Wharton's jelly for cell adhesion;

[0182] (ii) incubate the adherent cells obtained in step (i) in a medium containing platelet lysate.

[0183] The process includes the following steps:

[0184] (i) cultivate human umbilical cord tissue containing Wharton's jelly in a culture medium for cell adhesion;

[0185] (ii) incubate the adherent cells obtained in step (i) in a medium containing platelet lysate.

[0186] In one embodiment, said process comprises the following steps:

[0187] (i) to culture human umbilical cord tissue containing Wharton's jelly in a clinical-grade culture medium for cell adhesion;

[0188] (ii) incubate the adherent cells obtained in step (i) in a medium containing platelet lysate;

[0189] characterized in that said tissue comes from a mother meeting at least one of the following criteria: having received an administration of oxytocin during delivery, having given birth by induced labor, having given birth at term, not having experienced preeclampsia during pregnancy, whose child did not present neonatal disorders and having been exposed to tobacco smoke during pregnancy.

[0190] The aforementioned selection criterion for umbilical cords related to the mother allows for obtaining CSM-GW with better proliferation properties.

[0191] According to the invention, human umbilical cord tissues are collected at maternity wards immediately after delivery from a mother meeting the above criteria.

[0192] In one embodiment, human umbilical cords are severed and cultured. In another embodiment, the umbilical arteries and vein are removed before culturing the umbilical cords. In yet another embodiment, the umbilical arteries and vein are not removed before culturing the umbilical cords.

[0193] In one embodiment, umbilical cords are washed before being cultured. In another embodiment, the umbilical cords are rinsed in an a-MEM solution, preferably a clinical-grade a-MEM solution. In another embodiment, the a-MEM solution, preferably a clinical-grade a-MEM solution, further comprises at least one antibiotic and / or at least one antifungal. In one embodiment, said at least one antibiotic is selected from gentamicin, arnoxicillin, and vancomycin. In another embodiment, said at least one antifungal is amphotericin B.

[0194] In one embodiment, the umbilical cords are rinsed before being cultured. In another embodiment, the umbilical cords are rinsed in a PBS solution.

[0195] In one embodiment, the culture of human umbilical cord tissue containing Wharton's jelly for cell adhesion is a dry culture. In one embodiment, the culture medium, preferably the clinical-grade culture medium for cell adhesion used in step (i), is an α-MEM medium.

[0196] In one embodiment, the platelet lysate-containing medium used in step (ii) is an α-MEM medium. In another embodiment, the platelet lysate-containing medium used in step (ii) further comprises 1% to 20% platelet lysate, preferably 1% to 15%, preferably 1% to 10%, preferably 2.5% to 7.5%, preferably about 5% platelet lysate. In another embodiment, the platelet lysate-containing medium used in step (ii) further comprises at least one antibiotic. In one embodiment, said at least one antibiotic is gentamicin. In another embodiment, the platelet lysate-containing medium used in step (ii) further comprises heparin.

[0197] In one embodiment, step (ii) of the process of the invention is carried out under hypoxic conditions, preferably at approximately 5% CO₂ / 5% Ch. In another embodiment, step (ii) of the process of the invention is carried out at 37°C. In another embodiment, during the implementation of step (ii) of the process of the invention, the cells are cultured to 80% + / -10% confluence.

[0198] Advantageously, step (ii) of said process is repeated several times, in particular 2 or 3 times. In the present invention, each repetition of step (ii) of the process shall be referred to as a "pass". For example, the first implementation of step (ii) of the process corresponds to pass PO; its first repetition corresponds to the first pass (or pass PI); its second repetition corresponds to the second pass (or pass P2); etc.

[0199] According to the invention, when step (ii) is repeated, after obtaining 80% + / - 10% confluence, the cells are detached by any technique known to those skilled in the art, in particular by the action of clinical grade trypsin.

[0200] According to the invention, the cells harvested at the end of step (ii) are cryopreserved at about -20°C, preferably at about -80°C, preferably between -150°C and -196°C, preferably at about -150°C.

[0201] BRIEF DESCRIPTION OF THE FIGURES

[0202] The present invention is illustrated in more detail in the figures and examples described below.

[0203] Figures 1A and 1B: These figures represent the mean number of CFUs per milligram of spleen in anaerobic (Figure 1A) and aerobic (Figure 1B) conditions, respectively. Results are presented as mean ± SEM. n = 7–12 mice per group. *p < 0.05 CSM versus PBS.

[0204] Figures 2A and 2B: These figures represent the number of CFUs in anaerobic (Figure 2A) and aerobic (Figure 2B) conditions, respectively, per ml of blood. Results are presented as mean ± SEM. n = 7–12 mice per group. *p < 0.05 CSM versus PBS.

[0205] Figures 3A, 3B, 3C, 3D: These figures represent neutrophil kinetics in the lung (Figure 3A), spleen (Figure 3B), liver (Figure 3C), and femur (Figure 3D), respectively. Results are presented as mean ± SEM. n = 4–6 mice per group. *p < 0.05 CSM versus PBS; **p < 0.01 CSM versus PBS; ***p < 0.001 CSM versus PBS.

[0206] Figures 4A, 4B, 4C, and 4D: These figures represent the kinetics of monocytes in the lung (Figure 4A), femur (Figure 4B), and inflammatory monocytes in the lung (Figure 4C) and femur (Figure 4D), respectively. Results are presented as mean ± SEM. n = 4–6 mice per group. *p < 0.05 CSM versus PBS; **p < 0.01 CSM versus PBS; #p < 0.01 CSM-MO versus CSM-GW.

[0207] Figure 5: This figure shows the survival rate after cecal ligation and perforation in mice treated with CSM-GW, CSM-MO, and untreated mice. The results are presented as Kaplan-Meier curves. n = 18–25 mice per group. *p < 0.05 CSM versus PBS.

[0208] Figures 6A, 6B, 6C, 6D, 6E, 6F, 6G, and 6H: These figures show the impact of CSM-GW treatment on sepsis in pigs compared to untreated pigs. Figure 6A compares venous oxygen saturation over time in the CSM-GW treatment group and the untreated group (p < 0.01 CSM-GW vs. control). Figure 6B compares the decrease in mean arterial pressure over time in the CSM-GW treatment group and the untreated group (p < 0.01 CSM-GW vs. control). Figure 6C compares the administered norepinephrine level over time in the CSM-GW treatment group and the untreated group (p < 0.0001 CSM-GW vs. control). Figure 6D compares plasma creatinine levels over time in the CSM-GW treatment group and the untreated group (**p<0.01 CSM-GW versus control).Figure 6E compares diuresis in the CSM-GW treatment group and the untreated group. Figure 6F compares lactate levels over time in the CSM-GW treatment group and the untreated group (p < 0.01 CSM-GW vs. control). Figure 6G compares the PaO2 / FiO2 ratio over time in the CSM-GW treatment group and the untreated group (p < 0.5 CSM-GW vs. control). Figure 6H compares animal survival over time in the CSM-GW treatment group and the untreated group.

[0209] Figures 7A, 7B: These figures show the impact of obstetric factors on cell proliferation (Figures 7A and 7B). Doubling time is inversely proportional to proliferation. *: p < 0.05 in bivariate regression, **: p < 0.05 in multivariate regression.

[0210] EXAMPLES

[0211] Example 1: Production of CSM-GWs, Isolation of CSM-GWs

[0212] Human umbilical cords are collected at the Maternity Ward of the University Hospital of Nancy after informing the donor mothers and obtaining their written consent. The cords are placed in a collection medium and stored at 4°C. The collection medium, placed in a sterile container (e.g., Cryokits, Verreries Talançonnaises), is composed of phosphate-buffered saline (PBS, Macopharma, BC0120020) to maintain a pH close to 7, heparin to prevent residual blood from clotting, and an antibiotic (gentamicin). The cord can be stored for 24 hours at 4°C in the transport medium after collection.

[0213] Upon receipt, umbilical cords are immersed for one hour at room temperature in an antibiotic-antifungal solution composed of gentamicin, amoxicillin, vancomycin, and amphotericin B in clinical-grade a-MEM culture medium (Macopharma, BC0110010) with final concentrations of 0.5 g / L, 1 g / L, 1 g / L, and 0.05 g / L, respectively. This bath reduces the risk of microbial growth during culture that could be secondary to contamination during collection. Once the cord is decontaminated, the vein and the external portion of the cord are washed with PBS. 10 mL of PBS are injected into the umbilical vein using a syringe to remove residual blood. The cord is then pre-cut into 5 cm pieces using a sterile scalpel, and thin cross-sections (2 to 3 mm thick) are made. These pieces are then placed in culture boxes with removable lids (TPP, 90552).The pieces are dry-adhered to the bottom of the plates for 15 minutes, then complete medium, composed of α-MEM supplemented with 5% platelet lysate (Macopharma, BCO 190020) in the presence of gentamicin and heparin, is added. Culture is carried out in a dedicated incubator at 37°C and 5% CO2 under hypoxic conditions (5% Cte). After 5 days, the culture medium is changed.

[0214] After approximately 10 days of culture, the CSM-GW cells will have migrated and adhered to the bottom of the culture dishes. The fragments are then removed using sterile forceps, and the dish is washed with PBS buffer. The cells are then cultured until confluence (>80%) between 15 days and 3 weeks (between day 14 and day 21).

[0215] CSM-GW Culture

[0216] After confluence is achieved, the cells are detached by the action of recombinant GMP trypsin (TrypLE, Invitrogen).

[0217] After washing, the cells are reseeded at lxlO3 cells per cm 2 in passage 1 in complete medium. A 2-tier culture unit of 1270 cm 2 A CellSTACK-type surface plate (CELLSTACK2, Macopharma) is used, along with a universal cap with a chimney (MPC Cap, Macopharma). The inoculation kit (BC0400011, Macopharma) includes the tubing system for adding the various medium components and cells, as well as the connectors for attaching it to the culture container.

[0218] The medium is changed once a week until confluence is achieved (>80%) (between days 7 and 8 after initiation of culture). For this, a medium change kit (BC0400021, Macopharma) is used, which includes the tubing system for adding the different medium components, a waste bag, and the connector for attaching it to the culture container, as well as a universal stopper with a chimney. However, most of the time, the PI (Plant Integrity) phase lasts one week, and a medium change is not necessary. Once confluence is achieved, the cells are cryopreserved in a master cell bank (MCB). The Food and Drug Administration (FDA) defines an MCB as a collection of cells of uniform composition, derived from a single tissue or cell, and stored in an aliquot under defined conditions.

[0219] Final collection of cells

[0220] After confluence is achieved at the end of the PI step, the cells are collected by the action of trypsin as described previously and then washed. Viability and cell count checks, as well as various PI controls, are performed (see table below). The cells are then frozen.

[0221] Conservation of MCB CSMs

[0222] The MSCs are stored in tanks (in nitrogen vapor) at -150°C with continuous recording of the temperature and nitrogen level. In addition, 5 to 10 sample tubes of MSCs are also frozen for quality control. Thawing of the MCB and washing of the MSCs

[0223] The MSCs are thawed and washed using Sepax (Biosafe) type technology and then reseeded in passage 2 and 3 according to the same protocol as for the PI.

[0224] Final collection of cells

[0225] After confluence is achieved at the end of the P3 passage, the cells are collected by the action of trypsin as described previously and then washed. Viability and cell count checks, as well as various end-of-production controls, are performed (see table below). The cells are then frozen in a Working Cell Bank (WCB). The Food and Drug Administration (FDA) defines a WCB as a collection of cells derived from one or more aliquots of a Microbiome Cell Bank (MCB). The MCB cells are expanded by serial subculture to the selected passage, after which the cells are combined, concentrated, and aliquoted. One or more aliquots of the WCB thus obtained can be used to produce a batch of the final product for use.

[0226] WCB CSM Conservation

[0227] The MSCs are stored in tanks (in nitrogen vapor) at -150°C with continuous recording of the temperature and nitrogen level. In addition, 5 to 10 sample tubes of MSCs are also frozen for quality control purposes.

[0228] Thawing, washing of WCB CSMs and transport of CSMs

[0229] The MSCs are thawed and washed using the Sepax (Biosafe) technique, then reconstituted in a 75 mL volume with 4% albumin and stored between 4 and 10°C. The MSCs are packaged in a bag (150 mL bag), labeled according to the regulatory procedure; this bag is placed in a secondary package (plastic bag), which is also labeled, and then in a transport container along with a temperature recorder, a validation certificate, an injection instructions, and a post-administration safety assessment form. Transport of the MSCs to the administration site is carried out by a carrier approved by the University Hospital.

[0230] Quality controls carried out during the production process

[0231] To ensure the sterility and safety of the product to be administered, product controls are performed at each stage of the culture process. All quality controls are listed in Table 1.

[0232]

[0233] Table 1 Cellular controls

[0234] Cellular numbering

[0235] On the starting product and at each cell collection stage (PO and P3), the cell count is determined using a hemocytometer after lysis of the red blood cells. Cell viability

[0236] On the starting product and at each stage of cell collection, viability is determined by flow cytometry by 7 AAD labeling at the end of PO, PI, P2, P3 must be >80%.

[0237] Cellular phenotype

[0238] The expression of surface markers characteristic of a cell type will be determined by flow cytometry after labeling the cells with monoclonal antibodies or corresponding isotypic controls.

[0239] At least 60% of the cells must express the following antigens: CD90, CD73, CD105, CD44, and 80% of the cells must not express: CD34, CD1b, CD19, CD45, HLA-DR (and / or IMF < 2 times the IMF of the isotype control). The CD106 marker must be identified in at least 10% of the cells.

[0240] CFU-F

[0241] Clonogenic capacity will be measured by culturing fibroblast progenitor cells (colony-forming unit fibroblasts: CFU-Fs). CFU-Fs (>50 cells) will be counted under an inverted microscope at 1x magnification, after fixation with methanol and Giemsa staining. Briefly, 2.5 and 5 x 10 2 cells are seeded in 5 mL of medium in a 25 cm³ flask 2The medium is completely renewed twice a week. The culture is stopped on day 10, fixed, and stained with Giemsa. Colonies with more than 50 cells are then counted. CFU-F culture is performed at the end of each passage. Immunological control

[0242] The lack of immunostimulatory capacity of MSCs is monitored. To this end, a mixed lymphocyte culture will be performed using the produced and irradiated MSCs as stimulant cells and mononuclear cells from two healthy controls. The immunostimulatory effect is assessed by measuring the stimulation index. These tests are carried out in the quality control area of ​​the Innovative Therapy Drugs Prepared on an Ad Hoc Basis (ITD-PP) department of the Cell Therapy and Tissue Bank Unit (UTCT) in Nancy. The immunomodulatory effect is assessed by adding MSCs as a third partner to a conventional mixed lymphocyte culture. Microbiological control

[0243] Donor infection marker checks

[0244] Viral genomic testing is performed concurrently on the donor for HIV, HBV, and HCV. Conforming infectious markers are: HIV: Combined p24 antigen test + HIV 1+2 antibodies: Negative. HIV PCR: Negative; HBV: HBsAg negative, anti-HBc negative, HBV PCR: Negative; HCV: Anti-HCV antibodies: Negative, HCV PCR: Negative; HTLV: Anti-HTLV I+II antibodies: Negative; Syphilis: Anti-TP antibodies: Negative; CMV, EBV, and Toxoplasmosis: IgM negative, IgG negative or positive.

[0245] Bacteriology

[0246] Microbiological testing is performed according to the recommendations of the French National Agency for Medicines and Health Products Safety (ANSM) for Cell Therapy Products (CTPs) using aerobic and anaerobic blood cultures performed with the Bactec technique. This testing is carried out at each culture stage. For thawed mesenchymal stem cells (MSCs), the results of post-injection tests must be negative. In the event of a positive microbiological test after reinjection, the clinician will be informed immediately, and the identification of the causative organism and the results of the antibiogram will be provided in accordance with the internal procedure of the MTI-PP department. Other tests

[0247] The absence of a transcript hTERT

[0248] The absence of telomerase activity is assessed by qRT-PCR in MSCs after culture (end of PI and P3). This control will be performed on a 10% cryopreserved aliquot 6CSM. Karyotype

[0249] It is performed on a fresh MSC cell sample obtained at the end of culture (end of PI and P3), before cryopreservation.

[0250] Endotoxin and mycoplasma testing

[0251] It is performed on a fresh or frozen MSC cell sample obtained at the end of culture (end of PI and P3), before cryopreservation.

[0252] CSM Administration

[0253] The cells thus prepared are administered to hospitalized patients in intensive care presenting with septic shock or sepsis at a dose of at least 1x10 6 / kg heterologous MSCs in 75 mL of 4% albumin, NaCl, ACD, infused over 30 minutes via a central venous line. The treatment is received and administered via a central venous line preferably within 10 hours following the thawing and washing of the WCB MSCs, and at the latest up to 24 hours following the thawing and washing of the WCB MSCs.

[0254] Murine model of septic shock. Septic shock was induced in immunocompetent C57BL / 6 mice by cecal ligation and perforation (CLP). This model, considered the best standard for murine septic shock, mimics human peritonitis.

[0255] After surgery, the mice were randomized into 3 groups: one group receiving 0.25xl0 6 GW human CSM, a group receiving 0.25xl0 6 Bone marrow stem cells (BMSCs) obtained by culture from a bone marrow sample of a healthy, consenting donor were compared to a control group receiving prothrombin complex (PBS). Both MSCs and PBS were administered intravenously 24 hours after the onset of septic shock via the retro-orbital sinus.

[0256] CSM-GW and CSM-MO are used immediately after thawing without prior re-culturing.

[0257] Example 2: Effect of GW-MSCs on septic shock in mice. Impact of GW-MSCs on bacteremia.

[0258] Protocol

[0259] Thawed human MSCs were used. Forty-eight hours after the onset of septic shock, or 24 hours after MSC administration, the mice were euthanized by lethal injection of anesthetic. The spleen and blood of the mice were collected and then inoculated to count the colony-forming units (CFUs).

[0260] The comparison of the different groups was performed using a Kruskal-Wallis test. Results

[0261] Two days after septic shock, only mice treated with CSM-GW showed a significant decrease in bacteremia and splenic CFU count. The mean number of CFU per milligram of spleen or blood was counted. Forty-eight hours after the CLP procedure, the CSM-GW-treated group had an average of 2.3 x 10⁻⁶ CFU. 3CFU in aerobic conditions per mg of spleen and 5.8.10 3 Anaerobic CFU per mg of spleen, while the control group and the MO CSM-treated group had a mean number of aerobic CFU per mg of spleen of 9.5 x 10,000 respectively. 4 and 5.5X10 3 and an average number of anaerobic CFU per mg of spleen of 1.3 x 10 5 and Ι,ΙΧΙΟ 4 (Figures 1A and 1B). The results obtained in the blood are similar (Figures 2A and 2B). The group treated with MSCs derived from Wharton's jelly showed an average of 6 x 10 3 CFU in aerobic conditions / mL of blood and 2.1X10 4 Anaerobic CFU / mL of blood, while the control group and the MO CSM-treated group had a mean aerobic CFU / mL of blood of 1.3 x 10⁻¹⁰, respectively. 7 and 1.1X10 5 and an average number of anaerobic CFU per mL of blood of 7.7 x 10 6 and 3.2X10 5 .

[0262] Conclusions

[0263] These results show for the first time an antibacterial action of MSCs from Wharton's jelly in septic shock when used just after thawing.

[0264] Impact of CMS-GW on cell influx within oreans

[0265] Protocol

[0266] Forty-eight hours or seven days after the induction of septic shock, mice were euthanized by intraperitoneal injection of pentobarbital, and their organs were harvested. The spleen and liver were ground up and filtered. Bone marrow was extracted from the femur by rapidly injecting 1 ml of PBS into the medullary cavity. The lungs were cut into small pieces and placed in 2 ml of collagenase for 45 minutes before being ground up and filtered. Cells extracted from the different organs were washed by centrifugation, and a cell count was performed.

[0267] The cells were labeled with 5με-, anti-CD45, CDllb, Ly6C, Ly6G antibodies in order to identify and quantify total monocytes, inflammatory and anti-inflammatory monocytes, and neutrophils within the different organs.

[0268] The same protocol was performed on healthy mice before the induction of septic shock.

[0269] The comparison of the different groups was performed using a 2-way ANOVA followed by a Tukey test. Statistical significance was accepted for p < 0.05.

[0270] Results

[0271] Two days after septic shock induction, control mice receiving intravenous saline had a significantly higher pulmonary neutrophil count than mice treated with mesenchymal stem cell injection (Figure 3A). On day 7, a significantly lower neutrophil accumulation was observed in the spleens and livers of mice treated with MSCs of MO and GW (p<0.001) (Figures 3B, 3C). Seven days after CLP, the neutrophil count in the femurs of treated animals was significantly lower compared to the control group (PBS vs MO p<0.05; PBS vs GW p<0.01) (Figure 3D).

[0272] Two days after induction of septic shock and 24 hours after injection of MSCs, the lungs of mice that received MO or GW MSCs contained a number of inflammatory monocytes ly6C highsignificantly less than in control mice (p<0.01) (Figure 4A). A significant decrease in the total number of monocytes (Figure 4B) and in the number of inflammatory monocytes ly6C high (Figure 4C) at J2 was also found in the femurs of mice treated with CSM-GW (p<0.05) compared to control mice, while no significant difference was noted between the control group and the CSM-MO group.

[0273] Seven days after the induction of septic shock, a number of inflammatory monocytes ly6C high significantly less was observed in the spleens of mice treated with CSM-GW compared to mice treated with CSM-MO (p<0.01) and with PBS (<0.05) (Figure 4D).

[0274] This study highlights that MSCs are capable of modulating leukocyte pinfiltrate in a polymicrobial sepsis model. It shows that the action of thawed GW-MSCs is not limited to the first 48 hours following injection, but that they are capable of having much later effects on cell recruitment during septic shock.

[0275] In addition to their delayed efficacy, MSCs also exhibit an early effect on leukocyte trafficking. The results analyzed above show that MSCs reduce neutrophil influx into the lungs 48 hours after the induction of septic shock.

[0276] These results show that GW-MSCs, like MO-MSCs, are capable of reducing the accumulation of neutrophils within organs, which are implicated in the development of organ failure associated with septic shock. Indeed, it is known that abnormal neutrophil accumulation can induce, on the one hand, vascular occlusion leading to tissue hypoxemia and hypoperfusion, and on the other hand, microcirculatory dysfunction through the massive release of reactive oxygen species. Furthermore, unlike MO-MSCs, GW-MSCs are able to reduce both the femoral production of total monocytes two days after septic shock and the production of pro-inflammatory monocytes, leading to less accumulation of this monocyte subpopulation in the spleens of mice.Given that septic shock alters monocyte properties, notably by increasing their production of reactive oxygen species and thus raising the SOFA (sepsis-related organ failure assessment) score (Martins et al., 2008), the reduction in total and proinflammatory monocyte accumulation and production induced by the injection of GW-MSCs may offer a significant benefit in the treatment of septic shock. Impact of GW-MSCs on survival.

[0277] Protocol

[0278] After inducing septic shock in C57B1 / 6 mice using CLP, 150 μL of NaCl was administered subcutaneously to facilitate postoperative fluid resuscitation. To more closely mimic clinical practice, all mice received an intraperitoneal dose of 50 μg / g body weight of imipenem every 12 hours (Alcayaga-Miranda et al., 2015).

[0279] A Wilcoxon test was performed. A significant difference was considered to be p < 0.05. Results

[0280] The survival rate is increased in mice treated with MSCs compared to control mice. A survival rate of 64% was found in control animals, while 83% of animals treated with MSC-MO and 87% of animals treated with MSC-GW survived (Figure 5).

[0281] CSM-GW shows better results in terms of survival in septic shock compared to CSM-MO.

[0282] Example 3: Study of the impact of CSM-GW on seosis

[0283] The efficacy of thawed clinical grade CSM-GW produced according to the method described in part 1.1-1.6 above is analyzed in the pig, which is the closest living relative to humans from a cardiovascular point of view.

[0284] Four hours after the induction of peritonitis in pigs, a dose of CSM-GW of lxl0 6 / kg was injected intravenously. The CSM-GW were produced in clinical grade and used immediately after thawing. The study, conducted during the 24 hours following the induction of peritonitis, was double-blind and performed in the presence of an experienced critical care physician. Consequently, management was identical to that of a patient with maintenance of blood volume and mean arterial pressure (>85 mmHg) by fluid resuscitation and norepinephrine (maximum 10 μg / kg / min) and adequate cardiac output (>21 / min / m²). 2 ) by dobutamine (maximum 20μg / kg / min).

[0285] This study shows that the administration of CSM-GW significantly improves venous oxygen saturation, demonstrating a better match between O2 supply and O2 consumption in treated animals (Figure 6A).

[0286] Administration of CSM-GW improves cardiovascular function, as evidenced by the improvement in mean arterial pressure (MAP) in treated animals and the delayed administration of norepinephrine (Figures 6B, 6C). Administration of CSM-GW also improves renal function: treated animals exhibited a smaller increase in creatinine and greater diuresis (Figures 6D, 6E).

[0287] Furthermore, intravenous injection of CSM-GW significantly reduces lactate levels, the production of which exceeds 2 mmol / 1 indicates tissue hypoxia (Figure 6F).

[0288] Intravenous injection of CSM-GW also increases the PaCh / FiCte ratio, which reflects the severity of acute respiratory distress syndrome (Figure 6G). Its increase in treated animals indicates less lung failure compared to untreated animals. This experiment observed an increase in survival of approximately 60% in treated animals compared to untreated animals (Figure 6H).

[0289] Example 4: Analysis of obstetric factors

[0290] Fifty umbilical cords were analyzed by combining different obstetric parameters. CSM-GW were isolated from these tissues according to the method described in section 1.1-6 above.

[0291] After data extraction, the 27 obstetric factors (14 related to the mother, 6 to the newborn, and 7 to delivery) were analyzed for correlations between these obstetric parameters and 8 biological indicators of cell proliferation (population doubling). Each variable underwent bivariate linear regression (BLR). Only factors showing a significant association at the 0.15 level in BLR were candidates for multivariate linear regression (MLR). The stepwise variable selection method was used with a model entry threshold of 0.1 and a model exit threshold of 0.05.

[0292] After multivariate linear regression analysis, oxytocin administration at the time of delivery showed a positive impact on GW-MSC proliferation by decreasing the doubling time (61.6 ± 5.2 h vs. 112.0 ± 19.5 h, p = 0.0159) during the first PI passage (Figure 7A). This oxytocin administration also resulted in a greater number of cells with a short doubling time (<100 hours) (57.9% vs. 25%, p = 0.0469). Considering only samples with a PI doubling time of less than 100 hours, several factors had a positive impact on doubling time: directed labor (34.2% vs 0%, p = 0.0185), number of weeks of amenorrhea at birth (39.85 vs 37.92, p = 0.0212), maternal smoking (42.1% vs 8.3%, p = 0.0313) and placental weight (552.24 vs 481.92, p = 0.0446).At the second passage (P2), birth weight, gestational age, placental weight, normal pregnancy, and absence of preeclampsia showed a positive impact on cell proliferation (Figure 7B). All these factors are linked to the concept of term birth and demonstrate that GW-MSCs from healthy, term newborns exhibit superior proliferative capacities. It was also observed that GW-MSCs from umbilical cords of mothers who smoked exhibit increased proliferative capacities. Identifying these factors that promote GW-MSC proliferation can aid in the selection of umbilical cords containing GW-MSCs with improved proliferative properties.

[0293] BIBLIOGRAPHIE Alcayaga-Miranda, F., Cuenca, J., Martin, A., Contreras, L., Figueroa, F.E., and Khoury, M. (2015). Combination therapy of menstrual derived mesenchymal stem cells and antibiotics améliorâtes survival in sepsis. Stem Cell Res. Ther. 6.

[0294] Chao, Y.-H., Wu, H.-P., Wu, K.-H., Tsai, Y.-G., Peng, C.-T., Lin, K.-C, Chao, W.-R., Lee, M.-S., and Fu, Y.-C. (2014a). An Increase in CD3+CD4+CD25+ Regulatory T CelLs after Administration of Umbilical Cord-Derived Mesenchymal Stem Cells during Sepsis. PLoS ONE 9, ell0338.

[0295] Chinnadurai, R., Copland, I. B., Garcia, M. A., Petersen, C. T., Lewis, C. N., Waller, E. K., Kirk, A. D., and Galipeau, J. (2016). Cryopreserved Mesenchymal Stromal Cells Are Susceptible to T-Cell Mediated Apoptosis Which Is Partly Rescued by IFNy Licensing. Stem Cells, 34(9), 2429-2442.

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Claims

DEMANDS 1. Thawed human mesenchymal stem cells (MSCs) from Wharton's jelly for use in the treatment of sepsis, in particular septic shock.

2. Thawed human MSCs from Wharton's jelly for use according to claim 1, characterized in that the expression level of at least one marker selected from CD90, CD73, CD105, CD29, CD44, CD146, CD166, HLA-ABC is at least 10% lower than the expression level of the same marker in fresh human MSCs from Wharton's jelly.

3. Thawed human MSCs from Wharton's jelly for use according to claim 1 or 2, characterized in that the expression level of the marker CD90 is at least 10% lower than the expression level of the marker CD90 in fresh human MSCs from Wharton's jelly. 4.Defrosted human MSCs from Wharton's jelly for use according to any one of claims 1 to 3, characterized in that said MSCs express at least one protein selected from the group comprising ACTB, ANXA1, CAPZB, LASP1, PRDX2, PRDX3, PSA3, RS12, and SYWC.

5. Defrosted human MSCs from Wharton's jelly for use according to any one of claims 1 to 4, characterized in that said MSCs do not substantially express at least one protein selected from the group comprising ACTS, ALIBI, ANX10, GBB1, GBB2, GPRIN1, DTNA, MIP01, PSB3, and PSDE.

6. Thawed human MSCs from Wharton's jelly for use according to any one of claims 1 to 5, characterized in that said MSCs, under in vitro and / or non-inflammatory conditions, secrete at least one growth factor selected from BMP-7, IGFBP-1, insulin, FGF-7, NT-4 and VEGF-D.

7. Thawed human MSCs from Wharton's jelly for use according to any one of claims 1 to 6, characterized in that said MSCs, under in vivo and / or inflammatory conditions, secrete at least one growth factor selected from BMP-7 and TGFP3.

8. Thawed human MSCs from Wharton's jelly for use according to any one of claims 1 to 7, characterized in that said MSCs, under in vivo and / or inflammatory conditions, do not substantially secrete IGFBP-1.

9. Thawed human MSCs derived from Wharton's jelly for use according to any one of claims 1 to 8, characterized in that said MSCs secrete at least 1.2 times more VEGF than fresh human MSCs from Wharton's jelly.

10. Thawed human MSCs from Wharton's jelly for use according to any one of claims 1 to 9, characterized in that said MSCs induce an increase in serum VEGF concentration in the patient of at least 5% compared to fresh human MSCs from Wharton's jelly.

11. Thawed human MSCs derived from Wharton's jelly for use according to any one of claims 1 to 10, characterized in that said cells are derived from human umbilical cord tissue from a mother meeting at least one of the following criteria: having received oxytocin during delivery, having delivered by induced labor, having delivered at term, not having experienced preeclampsia during pregnancy, whose child did not experience neonatal disorders, and having been exposed to tobacco smoke during pregnancy.

12. Thawed human MSCs from Wharton's jelly for use according to any one of claims 1 to 11, characterized in that said MSCs are clinical grade cells.

13. Thawed human mesenchymal stem cells (MSCs) from Wharton's jelly for use according to any one of claims 1 to 12, characterized in that said cells are directly obtained from thawing without re-culture after thawing.

14. A pharmaceutical composition comprising thawed human mesenchymal stem cells from Wharton's jelly according to any one of claims 1 to 13 and a pharmaceutically acceptable excipient, for use in the treatment of sepsis.

15. A process for preparing clinical-grade mesenchymal stem cells from Wharton's jelly, comprising the following steps: (i) to culture human umbilical cord tissue containing Wharton's jelly in a clinical-grade culture medium for cell adhesion; (ii) incubate the adherent cells in a medium containing platelet lysate, characterized in that said tissue is derived from a mother meeting at least one of the following criteria: having received an administration of oxytocin during delivery, having given birth by induced labor, having given birth at term, not having experienced preeclampsia during pregnancy, whose child did not experience neonatal disorders and having been exposed to tobacco smoke during pregnancy.

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