Activated mesenchymal stromal cells

Abstract

The invention relates to a method of producing activated mesenchymal stromal cells (MSC), a composition comprising activated MSC, and use of said composition as a medicament, in particular for treatment of inflammatory conditions.

Description

[0001] Activated Mesenchymal Stromal Cells

[0002] Field of the invention

[0003] The invention relates to a method of producing activated mesenchymal stromal cells (MSC), a composition comprising activated MSC, and use of said composition as a medicament, in particular for treatment of inflammatory conditions.

[0004] Background

[0005] The placenta, a readily available and abundant source of stem cells, often discarded as medical waste, holds great promise as an attractive cell source for medical purposes.

[0006] There is considerable interest in the identification, isolation and production of human stem cells. Human stem cells are totipotent or multipotent progenitor cells capable of generating a variety of mature human cell lines. This ability serves as the basis for cell differentiation and specialization required for organ and tissue development.

[0007] The recent successful transplantation of such stem cells has provided a new clinical tool to reconstitute and I or replenish the bone marrow after exposure to diseased bone marrow resection, toxic chemicals and I or radiation.

[0008] Furthermore, there is evidence that stem cells can be used to repopulate many, if not all, tissues and restore physiological and anatomical functionality. The application of stem cells to tissue engineering, gene therapy delivery and cell therapy is also rapidly progressing.

[0009] A number of different types of mammalian stem cells have been characterized. For example, embryonic stem cells, embryonic germ cells, adult stem cells, or other committed stem cells or progenitor cells are known. Some stem cells are not only isolated and characterized, but are also cultured under conditions that allow differentiation to a limited extent. However, the basic problem remains that it is almost impossible to obtain a sufficient amount and population of human stem cells capable of differentiating into any cell type. The supply ofstem cells is extremely scarce. These are important for the treatment of a wide variety of acute inflammatory diseases.

[0010] An obstacle to realization of the therapeutic potential of stem cell technology has been difficulty in obtaining sufficient numbers of human stem cells. One source of stem cells is embryonic or fetal tissue. Embryonic stem and progenitor cells have been isolated from a number of mammalian species, including humans. The derivation of stem cells from embryonic or fetal sources, however, has raised many ethical and moral issues.

[0011] Stem cells also have been isolated from adult tissues. Methods for isolation of stem cells from adult sources often yield only limited quantities of cells and / or cells having limited ability to differentiate.

[0012] Obtaining a sufficient number of human stem cells has been difficult for several reasons. First, the separation of a normal population of stem cells in adult tissue is technically difficult and costly, in part because only a very limited amount of blood or tissue is observed. Second, the acquisition of these cells from fetal tissue, including embryos or miscarriages, has caused religious and ethical problems. Widespread support for the idea that human embryos and fetuses are independent lives has led to government regulations on the use of these sources for all uses, including medical research. Thus, alternative sources that do not require the use of cells obtained from embryonic or fetal tissue are essential to advance the use of stem cells in the clinic.

[0013] However, the supply is limited because there are very few possible alternative sources of stem cells, especially human stem cells. In addition, recovering sufficient amounts of stem cells from alternative sources for therapeutic and research purposes includes harvesting cells or tissues from donor subjects or patients, in vitro cell culture and I or proliferation, incisions, etc. Generally, it is complicated.

[0014] For example, Caplan et al. (1) discloses a bone marrow-derived human mesenchymal stem cell (hMSC) composition that serves as a precursor of a mesenchymal stem cell lineage, doing. Caplan et al. disclose that hMSCs are identified by specific cell surface markers identified with monoclonal antibodies. Positive selection of adherent bone marrow or periosteal cells thatdo not contain markers associated with either hematopoietic cells or differentiated mesenchymal stem cells results in uniform hMSC compositions. These isolated mesenchymal stem cell populations display the characteristics of the epitopes associated with mesenchymal stem cells and are capable of regenerating in culture without differentiation and are therefore induced or damaged in vitro When placed in vivo at a tissue site, it has the ability to differentiate into a specific mesenchymal lineage. However, such methods have the disadvantage that MSCs must be isolated after harvesting bone marrow or periosteal cells from a donor.

[0015] Postpartum tissues have generated interest as an alternative source for human stem cells. For example, methods for recovery of stem cells by perfusion of the placenta or collection from umbilical cord blood have been described. A limitation of stem cell procurement from these methods has been an inadequate volume of cord blood or quantity of cells obtained.

[0016] Hu et al. (2) isolated, cultured and cryopreserved for future use. Disclosed are human amnion epithelial cells derived from the placenta at delivery, either conserved or induced to differentiate. According to Hu et al., The placenta is collected immediately after delivery and the amniotic membrane is separated from the chorion, for example, by incision. The amniotic epithelial cells are then isolated from the amniotic membrane according to standard isolation techniques. The disclosed cells can be cultured in various media, grown in culture, cryopreserved, or induced to differentiate. Hu et al. disclose that amniotic epithelial cells are pluripotent (and possibly pluripotent) and can differentiate into epithelial tissues such as corneal surface epithelium or vaginal epithelium. However, the disadvantages of such methods are that they require a lot of effort and the yield of stem cells is very low. For example, to obtain a sufficient number of stem cells for typical therapeutic or research purposes, the amnion epithelial cells must first be isolated from the amnion by incision and cell separation techniques, and then cultured and expanded in vitro.

[0017] The currently available methods for ex vivo expansion of cell populations are also labor intensive. For example, Kraus et al. (3) allows for the selectivegrowth of a predetermined target cell population by: Discloses: after introducing a starting sample of cells from umbilical cord blood or peripheral blood into growth medium to divide the cells of the target cell population, the cells in the growth medium are bound to binding molecules having specific affinity. By contacting with a selection element containing (a monoclonal antibody for CD34), cells of a predetermined target population can be selected from cells in the growth medium.

[0018] Naughton et al. (4) is a stem cell or progenitor cell (eg, umbilical cord cell, Tissue culture in which placental cells, mesenchymal stem cells or stromal cells such as those derived from fetal cells) are grown on a three-dimensional support rather than a two-dimensional monolayer in a culture vessel such as a flask or dish.

[0019] Stem cells are extremely in short supply due to the limited collection and use of stem cells and the generally insufficient number of cells recovered from placenta. There is an urgent need for a source that makes a large number of human stem cells readily available for a variety of treatments and other medically relevant purposes. The present invention seeks to eliminate this demand and others.Brief description of the invention

[0020] In one aspect it is provided a method for producing a plurality of activated mesenchymal stem cells (MSC), the method comprising the steps:

[0021] i) providing a sample comprising placenta tissue derived MSC, preferably obtained from the choriotrophoblastic region of the placenta;

[0022] ii) initiating a cell culture comprising the MSC from step;

[0023] iii) adding microscaffolds to the culture comprising the MSC;

[0024] iv) allowing the MSC to adhere to the microscaffolds;

[0025] v) culturing the MSC adhered to the microscaffolds in dynamic suspension in a bioreactor under hypoxic conditions for at least 6 days, preferably at least 7 days, more preferably at least 9 days, most preferably at least 11 days;

[0026] vi) optionally repeating steps iii), iv) and v) at least once, preferably at least twice, more preferably twice;

[0027] vii) culturing the MSC for at least 16 hours, preferably at least 20 hours, more preferably at least 24 hours in the presence of tumor necrosis factor-a, preferably in a concentration of 10 ng / ml, interferon-y, preferably in a concentration of 10 ng / ml, and stromal derived factor 1 , preferably in a concentration of 100 ng / ml, such that a plurality of activated MSC is obtained;

[0028] viii) harvesting the plurality of activated MSC.

[0029] In another aspect, it is provided a method for producing a plurality of activated mesenchymal stem cells (MSC), the method comprising the steps:

[0030] i) providing a sample comprising placenta tissue derived fibroblast-like cells, preferably obtained from the choriotrophoblastic region of the placenta;

[0031] ii) culturing the placenta tissue derived fibroblast-like cells under hypoxic conditions in a 2D cell factory for at least 8 days in the presence of tumor necrosis factor-a, interferon-y, and stromal derived factor 1 , thereby obtaining MSC;

[0032] iii) selecting and harvesting MSC from the 2D cell factory, wherein MSC that show spindle shaped morphology are selected;

[0033] iv) initiating a cell culture comprising the selected MSC;

[0034] v) adding microscaffolds to the culture comprising the MSC;vi) allowing the MSC to adhere to the microscaffolds;

[0035] vii) culturing the selected and harvested MSC in a bioreactor under hypoxic conditions, preferably at an oxygen concentration of between 1 - 5%, preferably of between 2 -4 %, more preferably of between 2 - 3%, most preferably of 2% for at least 6 days, preferably at least 7 days;

[0036] viii culturing the MSC for at least 16 hours, preferably at least 20 hours, more preferably at least 24 hours in the presence of tumor necrosis factor-a, preferably in a concentration of 10 ng / ml, interferon-y, preferably in a concentration of 10 ng / ml, and stromal derived factor 1 , preferably in a concentration of 100 ng / ml, such that a plurality of activated MSC is obtained ix) harvesting the plurality of activated MSC obtained.

[0037] In one aspect it is provided a composition comprising a plurality of activated MSC, characterized in that the activated MSC express CD29, CD44, CD49d, CD54, CD73, CD90, CD105, CD106, VLA-4, ALCAM, CD18, CD49e, and MCAM, and expression of CXCR4, CCR2, CCR7, CXCR3, CCR5, CX3CR1, TLR2, TLR4, and HLA-G is increased by at least two-fold in the activated MSC when compared to MSC not cultured in the presence of tumor necrosis factorci, preferably in a concentration of 10 ng / ml, interferon-y, preferably in a concentration of 10 ng / ml, and stromal derived factor 1 , preferably in a concentration of 100 ng / ml.

[0038] In one aspect it is provided a composition comprising MSC-secreted exosomes, and optionally a plurality of MSC, characterized in that at least 10%, preferably at least 15%, more preferably at least 20% of the MSC-secreted exosomes is positive for CD9 or CD81.

[0039] In one aspect it is provided a composition comprising a plurality of MSC and / or MSC-secreted exosomes as described above, for use as a medicament.

[0040] Brief description of the drawings

[0041] Figure 1: Lymphocyte proliferation. CFSE histogram of (A) unstimulated and (B) Phytohaemagglutinin (PHA) stimulated PBMC samples.

[0042] Figure 2: Lymphocyte subtype proliferation, n = 3, single technical replicate. a-MSCs, activated mesenchymal stem cells; n-MSCs, naivemesenchymal stem cells; Unstirn, PHA unstimulated PBMCs; Control, PHA stimulated PBMCs. Group differences were assessed using the Kruskal-Wallis test, followed by Dunn’s post hoc test for pairwise comparisons. *p < 0.05 Figure 3: Changes in lymphocytes relative counts, n = 3, single technical replicate a-MSCs, activated mesenchymal stem cells; n-MSCs, naive mesenchymal stem cells; Unstirn, PHA unstimulated PBMCs; Control, PHA stimulated PBMCs. Group differences were assessed using the Kruskal-Wallis test, followed by Dunn’s post hoc test for pairwise comparisons. *p < 0.05, **p<0.01.

[0043] Figure 4: Cytokine concentrations in PHA stimulated PBMC samples. Cytokines measured: (A) IL-1 beta, (B) IL-10, (C) IL-13, (D) IL-17A, (E) IL-18, (F) IL-21, (G) IL-27, (H) IFN-gamma, (I) TNF-alpha. n = 3, measured in duplicates a-MSCs, activated mesenchymal stem cells; n-MSCs, naive mesenchymal stem cells; HS, hidradenitis suppurativa; IFN, interferon; IL, interleukin; TNF, tumor necrosis factor. Group differences were assessed using the Kruskal-Wallis test, followed by Dunn’s post hoc test for pairwise comparisons. Significant p-values (< 0.05) are indicated.

[0044] Figure 5: Cytokine concentrations in lesional skin biopsy samples.

[0045] Cytokines measured: (A) IL-1 beta, (B) IL-10, (C) IL-13, (D) IL-17A, (E) IL-18, (F) IL-21 , (G) IL-27, (H) IFN-gamma, (I) TNF-alpha. n = 3 for healthy subjects, n = 10 for adalimumab. a-MSCs, activated mesenchymal stem cells (n = 7); n-MSCs, naive mesenchymal stem cells (n = 10); HS, hidradenitis suppurativa (n = 10); IFN, interferon; IL, interleukin; TNF, tumor necrosis factor. Measured in duplicates. Group differences were assessed using the Kruskal-Wallis test, followed by Dunn’s post hoc test for pairwise comparisons. Significant p-values (< 0.05) are indicated.

[0046] Figure 6: Cytokine concentrations in perilesional skin biopsy samples.

[0047] Cytokines measured: (A) IL-1 beta, (B) IL-10, (C) IL-13, (D) IL-17A, (E) IL-18, (F) IL-21 , (G) IL-27, (H) IFN-gamma, (I) TNF-alpha. n = 3 for healthy subjects, n = 10 for adalimumab. a-MSCs, activated mesenchymal stem cells (n = 7); n-MSCs, naive mesenchymal stem cells (n = 10); HS, hidradenitis suppurativa (n = 10); IFN, interferon; IL, interleukin; TNF, tumor necrosis factor. Measured induplicates. Group differences were assessed using the Kruskal-Wallis test, followed by Dunn’s post hoc test for pairwise comparisons. Significant p-values (< 0.05) are indicated.

[0048] Detailed description

[0049] The invention provides for a robust and scalable process that incorporates both two-dimensional (2D) and bioreactor-based methodologies. Initially, MSCs are cultivated and selected in 2D cell factories to ensure optimal growth and phenotypic stability. These cells are subsequently transferred to a bioreactor system for large-scale expansion under controlled conditions.

[0050] In an alternative process, MSCs are entirely cultivated in a bioreactor-based system, eliminating the need for intermediate 2D culture.

[0051] In one embodiment, the invention provides a method for producing a plurality of activated mesenchymal stem cells (MSC), the method comprising the steps:

[0052] i) providing a sample comprising placenta tissue derived MSC, preferably obtained from the choriotrophoblastic region of the placenta;

[0053] ii) initiating a cell culture comprising the MSC;

[0054] iii) adding microscaffolds to the culture comprising the MSC;

[0055] iv) allowing the MSC to adhere to the microscaffolds;

[0056] v) culturing the MSC adhered to the microscaffolds in dynamic suspension in a bioreactor under hypoxic conditions for at least 6 days, preferably at least 7 days, more preferably at least 9 days, most preferably at least 11 days;

[0057] vi) optionally repeating steps iii), iv) and v) at least once, preferably at least twice, more preferably twice;

[0058] vii) culturing the MSC for at least 16 hours, preferably at least 20 hours, more preferably at least 24 hours in the presence of tumor necrosis factor-a, preferably in a concentration of 10 ng / ml, interferon-y, preferably in a concentration of 10 ng / ml, and stromal derived factor 1 , preferably in a concentration of 100 ng / ml, such that a plurality of activated MSC is obtained;

[0059] viii) harvesting the plurality of activated MSC.As described above in step vi) and in the Example section, it is advantageous to add fresh microscaffolds several times during the complete culturing process that can take up to 28 days or more. Preferably, fresh microscaffolds are thus added at the initiation of the culture at day 0, then freshly added after culturing for about 12 days, then again after culturing for an additional 10 days. This provides for sufficient substrate for the cells to adhere to when expanding. Alternatively, the invention provides for a method for producing a plurality of activated mesenchymal stem cells (MSC), the method comprising the steps: i) providing a sample comprising placenta tissue derived fibroblast-like cells, preferably obtained from the choriotrophoblastic region of the placenta;

[0060] ii) culturing the placenta tissue derived fibroblast-like cells under hypoxic conditions in a 2D cell factory for at least 8 days, preferably at least 12 days, more preferably at least 15 days, more preferably at least 20 days in the presence of tumor necrosis factor-a, interferon-y, and stromal derived factor 1 , thereby obtaining MSC;

[0061] iii) selecting and harvesting MSC from the 2D cell factory, wherein MSC that show spindle shaped morphology are selected;

[0062] iv) initiating a cell culture comprising the selected MSC;

[0063] v) adding microscaffolds to the culture comprising the MSC;

[0064] vi) allowing the MSC to adhere to the microscaffolds;

[0065] vii) culturing the selected and harvested MSC in a bioreactor under hypoxic conditions, preferably at an oxygen concentration of between 1 - 5%, preferably of between 2 -4 %, more preferably of between 2 - 3%, most preferably of 2% for at least 6 days, preferably at least 7 days;

[0066] viii culturing the MSC for at least 16 hours, preferably at least 20 hours, more preferably at least 24 hours in the presence of tumor necrosis factor-a (TNF-a), preferably in a concentration of 10 ng / ml, interferon-y (IFN-y), preferably in a concentration of 10 ng / ml, and stromal derived factor 1 (SDF-1), preferably in a concentration of 100 ng / ml, such that a plurality of activated MSC is obtained;;

[0067] ix) harvesting the plurality of activated MSC obtained.As described above under step iii), the selection process after the 2D culture is based on the morphology of the cells. In particular the cells should present with a spindle shaped morphology, preferably with a lenght-to-wide ratio of 3:1 to 5:1 as determined by microscopic image analysis. The cells may be selected based on further characteristics such as superior adherence to tissue culture plastic, and a doubling time of less than 24 hours under the defined culture conditions.

[0068] The cytokines used in the final stage of culturing have several effects on the MSC, rendering them activated MSC. TNF-a and IFN-y act in concert to upregulate key immunomodulatory molecules, including IDO (indoleamine 2,3-dioxygenase) and PGE2 (prostaglandin E2), amplifying the MSCs’ ability to suppress excessive inflammation in AKI. TNF-a and IFN-y also enhance the expression of VCAM-1 (vascular cell adhesion molecule-1) and ICAM-1 (intercellular adhesion molecule-1) on endothelial cells and integrins on MSCs, improving their adhesion to inflamed vasculature and facilitating transmigration to the injured renal tissue.

[0069] SDF-1 (also known as CXCL-12) binds to CXCR4 receptors on MSCs, enhancing their chemotaxis and migration toward the injured kidney, where SDF-1 gradients are naturally elevated. The SDF-1 / CXCR4 axis not only directs MSCs to the site of injury but also promotes their retention within renal tissue, ensuring sustained therapeutic effects. SDF-1 further stimulates the secretion of angiogenic factors from MSCs, promoting vascular repair and epithelial regeneration

[0070] The cocktail of SDF-1 , TNF-a and IFN-y activates complementary molecular pathways, including PI3K / Akt and NF-KB, to enhance MSC survival, reduce oxidative stress, and promote anti-fibrotic effects in the kidney. Combined, TNF-a, IFN-y, and SDF-1 prime MSCs to release a targeted secretome enriched with anti-inflammatory cytokines, pro-regenerative growth factors, and extracellular vesicles carrying therapeutic microRNAs.

[0071] The activation of the MSC has herapeutic advantages, e.g., in acute kidney injury (AKI), such as enhanced homing and retention: By integrating homingenhancing SDF-1 with priming cytokines TNF-a and IFN-y, this cocktailensures higher MSC delivery to injured renal tissues and sustained functional activity. The cytokine cocktail addresses the critical challenges of MSC therapy for AKI, including low cell retention, inadequate homing, and suboptimal therapeutic potency.

[0072] In a preferred embodiment, the microscaffolds are cationic charged crosslinked poysterene microscaffolds, preferably cross-linked dextran or gelatinous-suspension microscaffolds. These microscaffolds have excellent adherence characteristics for MSC.

[0073] Preferably, throughout the complete culturing period, the culture medium is free of added antibiotics, in particular the culture medium is free of penicillin and of streptomycin. The absence of antibiotics improves on the cell growth and differentiation of the MSC.

[0074] Now that the invention provides for a method for producing activated MSC, the invention further provides for a composition obtainable by a method according to the invention.

[0075] Further provided is a composition comprising a plurality of activated MSC, characterized in that the activated MSC express CD29, CD44, CD49d, CD54, CD73, CD90, CD105, CD106, VLA-4, ALCAM, CD18, CD49e, and MCAM, and expression of CXCR4, CCR2, CCR7, CXCR3, CCR5, CX3CR1, TLR2, TLR4, and HLA-G is increased by at least two-fold in the activated MSC when compared to MSC not cultured in the presence of tumor necrosis factor-a, preferably in a concentration of 10 ng / ml, interferon-y, preferably in a concentration of 10 ng / ml, and stromal derived factor 1 , preferably in a concentration of 100 ng / ml. In a preferred embodiment, the plurality of activated MSC is obtainable by a method according to the invention.

[0076] The secretome derived from mesenchymal stromal cells (MSCs) activated with tumor necrosis factor-a, preferably in a concentration of 10 ng / ml, interferon-y, preferably in a concentration of 10 ng / ml, and stromal derived factor 1 , preferably in a concentration of 100 ng / ml includes a well-defined profile of exosomes that can be used for the indications metioned further down below. The invention thus provides for a composition comprising an MSC secretome comprising MSC- derived exosomes, and optionally a plurality of MSC,characterized in at least 10%, preferably at least 15%, more preferably at least 20% of the MSC-secreted exosomes is positive for CD9 or CD81. In a preferred embodiment, the MSC secretome comprising MSC-derived exosomes, and the optional plurality of MSC are obtainable by a method according to the invention.

[0077] As the skilled person will appreciate, the secretome of MSC is the totallity of substances that are secreted by the MSC into the extracellular envirnment. The exosomes are part of the secretome and are small, membrane-bound vesicles that are released by cells. They contain various molecules such as proteins, lipids, and RNAs and can be used as cell-free therapeutics that convey healing properties of the underlying cells, in this case the MSC, by serving as cellular messengers.

[0078] The protein profile of the secretome preferably reveals a protein concentration ranging from 0.7-43 pg / L across measured samples, with total protein levels ranging from 4,760-38,578.8 pg. The RNA profile shows RNA concentrations ranging from 13.5-132 ng / pL and total RNA levels between 64.0-2,632.0 ng. The particle profile includes protein concentrations ranging from 3.64-16.77 pg / L, particle concentrations between 3.28 x 1010— 1.51 x 1011particles / mL, mean particle sizes of 120.1-132.3 nm, mode particle sizes of 70.9-90.2 nm, and particle numbers per pg of protein ranging from 4.8 x 107— 1.1 x 108.

[0079] The secretome is preferably enriched with microRNAs, with RNA concentrations ranging from 6.76-29.24 ng / pL. Normalized microRNA levels include miR-146b ranging from 0.74-1.19, miR-24 from 0.74-1.19, miR-124 from 0.74-1.17, miR-125 from 1.05-1.24, miR-149from 0.74-0.97, and miR-555 from 0.71-0.89.

[0080] The functional gene expression profile preferably highlights TNFAIP6 with ACT values ranging from 6.594-8.328 and fold changes of 1.158-3.327, demonstrating enhanced anti-inflammatory activity. TGFB1 preferably shows CT values from 2.361-4.649 and fold changes of 1.060-4.883, indicating strong pro-regenerative and anti-fibrotic signaling. VEGFA preferably exhibits ACT values of 3.692-5.240 and fold changes of 1.148-2.924, contributing to angiogenesis and vascular repair. COX2 / PTGS2 preferably displays ACTvalues ranging from 10.770-17.562 and fold changes from 0.064-7.126, highlighting its role in inflammation resolution pathways

[0081] The functional kynurenine concentration profile from absorbance spectroscopy preferably shows activated cells producing levels between 9.01-16.71 pg / mL, while non-activated cells produce significantly lower levels between 0.38-0.49 pg / mL. Results from the HPLC-MS assay confirm these findings, with activated cells producing kynurenine concentrations ranging from 6.69-11.74 pg / mL and non-activated cells producing concentrations from 0.34-0.38 pg / mL.

[0082] The activated MSC of the invention can be readily distinguished from other MSC by their activation profile, which is advantageous for the treating inflammotry conditions, listed further down below. The invention thus provides for a composition according to the invention, wherein the MSC express tumor necrosis factor alpha-induced protein 6 with a delta Ct relative to GAPDH of at least 4, preferably at least 5, more preferably at least 6, transforming growth factor beta 1 with a delta Ct relative to GAPDH of at least 2, vascular endothelial growth factor A with a delta Ct relative to GAPDH of at least 2, prefarably at least 3, more preferably at least 3.5, and / or cyclooxygenas-2 with a delta CT relative to GAPDH of at least 5, preferably at least 7, more preferably at least 9, most preferably at least 10.

[0083] Further provided is a composition according to the invention, wherein less than 1 % of the MSC express or secrete SCF, FLT-3, ALDH X, or IL-6 and / or wherein the composition is essentially free of SCF, FLT-3, ALDH X, and IL-6. Further provided is a composition according to the invention for use as a medicament, in particular for the treatment of an inflammatory condition. Also provided is a method of treating an inflammatory condition, the method comprising administering a composition according to the invention to a subject suffering from the inflammatory condition.

[0084] In a preferred embodiment, the inflammatory condition is selected from the group consisting of acute inflammation, acute kidney injury, acute lung injury, acute liver injury, acute pancreatitis, acute severe colitis, acute nodular flares in hidradenitis suppurativa, an autoimmune disorder, and a chronic degenerative disease. These diseases are explained in more detail below.Acute inflammation is an immediate adaptive response with limited specificity caused by several noxious stimuli, such as infection and tissue damage (tissue necrosis). The controlled inflammatory response is generally beneficial and can be seen clearly in protecting against infectious organisms, including Mycobacterium tuberculosis, protozoa, fungi, and other parasites. However, it can be detrimental if not regulated, as observed in septic shock. The inflammatory pathway involves inducers, sensors, mediators, and effector molecules.

[0085] The process is initiated in the presence of inducers, infectious organisms, or non-infectious stimuli such as foreign bodies and signals from necrotic cells or damaged tissues. This, in turn, activates sensors that are specialized molecules. The sensors then stimulate the mediators, which are endogenous chemicals that can induce pain, activate or inhibit inflammation and tissue repair, and activate the effectors, which are the tissues and cells. These players can act together and give rise to multiple alternative pathways in the inflammatory process, depending on the type of stimulus. The goal of the inflammatory process is to restore homeostasis regardless of the cause.

[0086] Acute Kidney Injury (AKI). AKI signifies a sudden and often temporary decline in kidney function, gauged by the glomerular filtration rate (GFR).

[0087] Intriguingly, measures such as blood urea nitrogen (BUN) and creatinine levels may remain in their standard brackets immediately after kidney distress. The primary indication of AKI is reduced urine production.

[0088] Currently, the treatment of acute kidney injury primarily involves ensuring adequate hydration, often through intravenous fluids. Maintaining balanced electrolyte and blood sugar levels is essential. Depending on the cause, specific medications may be prescribed to address the underlying issues, such as medicines to manage high blood pressure or to alleviate potential kidney toxins. In severe cases, dialysis is necessary to support the kidneys.

[0089] However, the described treatments are primarily aimed at alleviating the progression of kidney damage, with limited effectiveness in restoring the lost kidney function. Multiple cellular protective agents have been tested for treating acute kidney injury. However, many studies have not shown consistentsuccess, including specific receptor antagonists, channel blockers, and various antibodies. Direct interventions such as dialysis might only provide temporary relief without addressing the root cause of kidney injury.

[0090] Acute Lung Injury (ALI). ALI represents a rapid, frequently transient decline in lung functionality, which is often measured by gas exchange efficiency. Following initial lung distress, parameters such as arterial oxygen levels may remain within the standard limits. At times, the primary symptoms of ALI can be reduced oxygen saturation or difficulty breathing.

[0091] Currently, the treatment for acute lung injury primarily involves ensuring optimal oxygenation, frequently through supplemental oxygen or mechanical ventilation. Concurrently, it is crucial to maintain a balanced fluid and electrolyte status. Depending on the cause, specific medications may be administered to treat underlying conditions, such as drugs to manage inflammation or counteract potential lung toxins. More invasive respiratory support methods such as high-frequency ventilation may be employed in severe cases.

[0092] However, these treatments are mainly directed at alleviating the progression of lung damage with limited success in fully restoring lost lung function. Multiple therapeutic agents have been explored to treat acute lung injury (ALI).

[0093] However, many studies have not consistently shown its efficacy, including specific receptor antagonists, anti-inflammatory agents, and various antibodies. Interventions such as mechanical ventilation may only provide temporary assistance without confronting the fundamental cause of lung injury.

[0094] Acute Liver Injury (ALI). ALI denotes a swift, often temporary, decline in liver functionality, typically assessed by liver enzyme levels and other liver function tests. After the onset of liver distress, parameters such as bilirubin or alanine aminotransferase (ALT) levels may remain within the standard ranges.

[0095] Occasionally, the primary manifestation of ALI is jaundice or altered liver function.

[0096] The treatment of acute liver injury mainly centers on supporting liver function and preventing further damage, often through strict monitoring and specific medical interventions. Simultaneously, maintaining a balanced fluid,electrolyte, and nutrient status is imperative. Depending on the cause, specialized medications, such as drugs to manage inflammation or neutralize potential liver toxins, may be administered to address the underlying conditions. More intensive measures, such as liver dialysis, should be considered in severe cases.

[0097] However, the outlined treatments aim to mitigate the progression of liver damage, with limited effectiveness in fully recuperating lost liver function. A range of therapeutic agents has been investigated for the treatment of acute liver injury. Nonetheless, many have yet to uniformly exhibit success, encompassing specific enzyme inhibitors, anti-inflammatory drugs, and various antibodies. Direct measures, such as liver dialysis, offer only temporary relief without addressing the intrinsic cause of liver injury.

[0098] Acute Pancreatitis (AP). AP represents a rapid, occasionally transient deterioration in pancreatic function, usually evaluated by elevated levels of pancreatic enzymes such as amylase and lipase. Following the onset of pancreatic distress, the levels of these enzymes can markedly increase.

[0099] Sometimes, the primary manifestation of AP is abdominal pain or digestive disturbances.

[0100] Currently, the treatment for acute pancreatitis predominantly revolves around relieving the symptoms and supporting the pancreas, commonly via fasting to allow organ rest, pain management, and intravenous fluids. Concurrently, it is crucial to maintain a balance between hydration and electrolyte levels.

[0101] Depending on the cause, specific medications may be administered to address underlying issues, such as drugs to address inflammation or mitigate potential pancreatic toxins. In severe cases, surgical intervention or drainage procedures are necessary.

[0102] However, the described treatments aim to alleviate the immediate symptoms and progression of pancreatic inflammation, with limited success in ensuring the full recovery of pancreatic function. Numerous therapeutic agents and strategies have been explored for treating acute treatment. However, many have yet to be consistently proven effective, including specific enzyme modulators, anti-inflammatory agents, and other compounds. Directinterventions, such as surgical drainage, may only provide transient benefits without treating the root cause of pancreatic inflammation.

[0103] Acute severe Colitis (ASUC). ASLIC indicates a rapid, often transient, exacerbation of ulcerative colitis symptoms, typically assessed by clinical presentation, laboratory markers, and imaging. After the onset of a flare-up, parameters such as C-reactive protein (CRP) or fecal calprotectin levels may remain within the standard ranges. Occasionally, the primary manifestation of ASUC is severe diarrhea, bloody stools, or acute abdominal pain.

[0104] Currently, the treatment of acute severe ulcerative colitis predominantly focuses on alleviating symptoms and preventing complications, often through vigilant monitoring and targeted medical interventions. Concurrently, ensuring balanced fluid, electrolyte, and nutritional status is paramount. Depending on the cause and severity, specialized medications, such as corticosteroids, immunosuppressants, or biologies, may be administered to manage inflammation and underlying disease activity. Hospitalization and colectomy may be considered in extreme cases. However, these treatments mainly strive to control acute symptoms and progression of colitis. However, they may have limited effectiveness in achieving a prolonged remission. Various therapeutic agents and approaches have been explored for the treatment of ASUC.

[0105] However, many studies have not consistently shown promising results, including specific targeted therapies, anti-inflammatory agents, and other novel treatments. Direct measures, such as surgical intervention, might provide a definitive solution but come with challenges and consequences.

[0106] Acute Nodular Flares in Hidradenitis Suppurativa (HS). Acute nodular flares in HS signify a rapid, often temporary, intensification of the skin condition, typically identified by the appearance of painful nodules and other skin manifestations. After the onset of such a flare-up, markers such as lesion count or disease seventy remained within the standard parameters.

[0107] Occasionally, the primary manifestation can be sudden, severe pain or a sudden increase in nodule size and number.

[0108] Treating acute nodular flares in HS can alleviate symptoms and prevent complications, often through vigilant monitoring and targeted medicalintervention. Maintaining optimal skin hygiene and care is crucial. Depending on the severity of the flare and the underlying triggers, specialized medications, such as antibiotics or immunomodulators, may be prescribed to manage inflammation and bacterial overgrowth. In more severe cases, surgical intervention or drainage procedures may be considered.

[0109] However, these treatments are primarily aimed at controlling the acute symptoms of HS. However, these treatments may have limited success in preventing recurrence or progression. Various therapeutic agents and modalities have been explored for the management of acute flares in HS patients. However, many drugs, including specific topical treatments, systemic medications, and emerging biologies, are yet to consistently demonstrate efficacy. Direct interventions, such as surgery, might provide immediate relief, but may not address the root cause or prevent future flare-ups.

[0110] Autoimmune disorders. Most prevalent autoimmune disorders that may be treated with a composition according to the invention: rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, type 1 diabetes mellitus, Crohn’s disease, ulcerative colitis, psoriasis, Hashimoto’s thyroiditis, Graves’ disease, ankylosing spondylitis, vitiligo, Sjogren’s syndrome, celiac disease, alopecia areata. Less prevalent autoimmune disorders that may be treated with a composition according to the invention: autoimmune encephalitis, Behget’s disease, dermatomyositis, polymyositis, pemphigus vulgaris, bullous pemphigoid, idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, Addison’s disease, antiphospholipid syndrome, scleroderma, myasthenia gravis, Guillain-Barre syndrome, autoimmune hepatitis, ataxiatelangiectasia.

[0111] Chronic degenerative diseases. Most prevalent degenerative diseases that may be treated with a composition according to the invention: osteoarthritis, Alzheimer’s disease, Parkinson’s disease, chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, chronic liver disease, chronic kidney disease, heart failure, diabetic neuropathy, age-related macular degeneration, glaucoma, degenerative disc disease. Less prevalent degenerative diseases that may be treated with a composition according to the invention: Friedreich’sataxia, Duchenne muscular dystrophy, hereditary spastic paraplegia, Charcot-Marie-Tooth disease, Huntington’s disease, retinitis pigmentosa, mitochondrial myopathy, spinal muscular atrophy, myotonic dystrophy, vascular dementia, sarcopenia, peripheral artery disease, non-alcoholic steatohepatitis (in early stages), intervertebral disc degeneration.

[0112] The invention is exemplified in the following non-limiting examples.

[0113] EXAMPLES

[0114] Example 1 - Isolation of Placenta-Derived MSC

[0115] Isolation

[0116] o Source: Placental or other defined tissues.

[0117] o Spinner flask isolation with rotating impeller:

[0118] 1. Use of a closed, sterile spinner flask with a rotating impeller to mechanically dissociate placental tissue into cell suspensions.

[0119] 2. Optimized impeller speed to ensure gentle dissociation, preserving cell viability while minimizing enzymatic exposure.

[0120] Example 2 - Bead-to-Bead MSC Expansion and Activation Process

[0121] 1. Initial Seeding on Polystyrene Microscaffolds (P0)

[0122] At passage 0 (P0), mesenchymal stromal cells (MSCs) are seeded onto polystyrene-based microscaffolds to facilitate cell attachment, proliferation, and expansion. Polystyrene was selected as the substrate due to its biocompatibility and ability to support adherent cell growth. Cultures are maintained under defined conditions for 12 days, during which the following parameters are routinely monitored:

[0123] • Cell count: Quantified to assess proliferation.

[0124] • Viability: Determined via live / dead assays to ensure healthy expansion.

[0125] • Confluence: Observed microscopically to evaluate the degree of surface coverage.

[0126] • Metabolic activity: Glucose consumption and lactate production are measured as indicators of cellular activity.

[0127] • Dissolved oxygen (DO): Maintained at optimal levels to ensure aerobic respiration and viability.2. Initial Addition of Fresh Microscaffolds (P1)

[0128] At P1 , 20% of MSC-populated microscaffolds from P0 are combined with 80% fresh, unpopulated microscaffolds. This facilitates the migration of MSCs from the previously colonized microscaffolds onto newly introduced surfaces. The migration is driven by the availability of additional substrate area and the natural motility of MSCs. Cultures are maintained for an additional 10 days to allow sufficient migration and proliferation.

[0129] During this phase, the same monitoring parameters — cell count, viability, confluence, glucose, lactate, and DO — are tracked to ensure optimal conditions and to determine the timing of subsequent steps.

[0130] 3. Second Fresh Microscaffolds Addition (P2)

[0131] The bead-to-bead transfer process is repeated at P2 by introducing another 80% fresh microscaffolds to the culture system. These fresh microscaffolds are mixed with 20% of the MSC-populated microscaffolds from P1. The migration and proliferation of MSCs onto the newly added microscaffolds further expand the cell population.

[0132] This step is maintained for 8 days under the same monitored parametersm after which harvesting is carried out.

[0133] Example 3 - Dual-Phase Cultivation Method

[0134] 1. Two-Dimensional (2d) Cell Factory Selection Methodology Selection and cultivation:

[0135] This approach goes beyond traditional in vitro culturing by focusing on monitoring and selecting a specific subset of MSCs within the culture. Cells are selected in 2D cell factories, where their ability to adhere to the culture surface and exhibit the characteristic spindle-shaped morphology is carefully observed and assessed.

[0136] Continuous monitoring allows for the identification and expansion of MSCs that demonstrate superior adherence properties and morphological potential, ensuring a homogenous and high-quality population for subsequent expansion.

[0137] Advantages:Enables precise selection of a specific subset of cells with consistent morphology and adherence traits, improving the overall therapeutic quality of the MSC composition.

[0138] Provides a controlled environment for rigorous monitoring and quality control, ensuring phenotypic stability at early stages of production.

[0139] 2. Bioreactor-Based Large-Scale Expansion

[0140] Process transition:

[0141] Once cells are selected in the 2d stage, they are transferred to a bioreactor system for large-scale expansion. The bioreactor provides a dynamic, three-dimensional culture environment under controlled conditions.

[0142] Bioreactor conditions:

[0143] The bioreactor is equipped with automated monitoring and feedback systems to maintain stable culture conditions.

[0144] Expansion and Monitoring:

[0145] o Partial conditioned media enriched with conditioned exosomes, cytokines, use in upstream process.

[0146] o Culturing under hypoxic conditions (2% O2) in chemically defined media.

[0147] o Stirred-tank or perfusion bioreactors with real-time monitoring of cell viability, growth rate, and metabolic activity.

[0148] This step supports high-density cell culture while retaining the therapeutic properties of the mses.

[0149] Advantages:

[0150] Enables large-scale production while maintaining cell quality. Reduces the risk of contamination due to the closed system. Provides scalability for clinical and commercial applications.

[0151] Example 4 - Cytokine-Based Activation

[0152] At the conclusion of the expansion process of both Example 2 and Example 3, MSCs are activated using a defined cytokine cocktail designed to prime the cells for enhanced therapeutic functionality. The cytokine cocktail comprises:

[0153] • Tumor Necrosis Factor-Alpha (TNF-a): Enhances immunomodulatory properties.• Interferon-Gamma (IFN-y): Induces the expression of anti-inflammatory molecules and immune-modulatory mediators.

[0154] • Stromal Cell-Derived Factor-1 (SDF-1): Promotes upregulation of CXCR4, improving the homing capacity of MSCs.

[0155] Activation is performed over 24 hours, during which cells remain in culture with cytokine-supplemented media.

[0156] Example 5 - Validation of Activation

[0157] The activation process is confirmed by analyzing the expression of functional markers that indicate the therapeutic readiness of the MSCs:

[0158] • Indoleamine 2,3-Dioxygenase (IDO): A key marker of immunomodulatory activity.

[0159] • HLA-G: Reflects the immunosuppressive and immune-tolerant phenotype of MSCs.

[0160] • CXCR4: Demonstrates enhanced homing potential, critical for sitespecific therapeutic efficacy.

[0161] Marker expression is validated via flow cytometry or equivalent immunophenotyping methods, ensuring the consistency and functional readiness of the MSCs for downstream applications.

[0162] 1. Core Identity Markers:

[0163] o At least 95% of the MSCs express CD73, CD90, and CD105, defining the cells as mesenchymal stromal cells according to ISCT criteria.

[0164] 2. Baseline Expression of Adhesion and Chemokine Markers:

[0165] o At least 85% of the MSCs express adhesion-related markers, including: CD29, CD44, CD49d, CD54, CD106, VLA-4, ALCAM, CD18, CD49e, and MCAM.

[0166] 3. Post-Activation Functional Upregulation:

[0167] o Activation with TNF-a (10 ng / mL), IFN-y (20 ng / mL), and SDF-1 (100 ng / mL) for 24 hours results in functional upregulation of specific markers, including:

[0168] ■ CXCR4: >3-fold increase, demonstrating enhanced homing potential.■ CCR2, CCR5, CCR7, CXCR3, CX3CR1 : >2-fold increase, reflecting improved migration and immunomodulatory properties.

[0169] ■ TLR2 and TLR4: >2-fold increase, indicating enhanced responsiveness to inflammatory stimuli.

[0170] ■ HLA-G: >2-fold increase, associated with elevated immunosuppressive capacity.

[0171] 4. Functional Validation:

[0172] o Migration: MSCs demonstrate >50% increased migration toward an SDF-1 gradient compared to non-activated cells in a transwell assay, correlating with CXCR4, CCR5, and CXCR3 expression. o Adhesion: Enhanced adhesion to fibronectin or collagen-coated surfaces, with >25% greater adhesion compared to non-activated cells, linked to VLA-4, CD49d, and ALCAM.

[0173] o Immunomodulation: Suppression of T-cell proliferation by >70%, correlating with upregulated HLA-G, TLR2, and TLR4. Marker-Specific Contributions

[0174] 1. Activation with TNF-a (10 ng / mL), IFN-y (20 ng / mL), and SDF-1 (100 ng / mL) for 24 hours results in functional upregulation of the following markers:

[0175] 2. Core identity markers:

[0176] o CD73, CD90, CD105: Constitutively expressed by >95% of MSCs. These markers do not show significant activationdependent changes.

[0177] 3. Adhesion and extracellular matrix interaction:

[0178] o CD29, CD44, CD49d, CD54, CD106, VLA-4, ALCAM, CD18, CD49e, MCAM: These markers facilitate adhesion and interaction with extracellular matrix components. Their expression is typically high (>85%), with minor upregulation (<20%) after activation, reflecting enhanced functionality.

[0179] 4. Chemokine Receptors (migration and homing):O CXCR4, CCR2, CCR5, CCR7, CXCR3, CX3CR1: These receptors are crucial for migration. Activation results in >2 -fold upregulation, particularly for CXCR4, which interacts with SDF-1 to direct MSC homing to injury sites.

[0180] 5. Inflammatory responsiveness:

[0181] o TLR2, TLR4: Toll-like receptors are moderately expressed in unactivated MSCs but show >2-fold upregulation post-activation with TNF-a and IFN-y, reflecting improved responsiveness to inflammatory environments.

[0182] 6. Immunomodulation:

[0183] o HLA-G: Baseline expression is moderate (30-40%) but increases to >80% after activation, enhancing immunosuppressive properties critical for therapeutic applications.

[0184] 7. Functional Validation:

[0185] o The MSCs demonstrate functional capabilities associated with the upregulated markers, including:

[0186] ■ Immunomodulation: >70% suppression of T-cell proliferation in co-culture assays, correlating with upregulated HLA-G expression.]

[0187] Example 6 - Analysis of the Secretome

[0188] The secretome derived from mesenchymal stromal cells (MSCs) activated with TNF-a, IFN-y, and SDF-1 includes a well-defined profile of exosomes enriched with bioactive molecules, proteins, and microRNAs (miRNAs). Secretome encapsulates the core therapeutic mechanisms of the cells in a bioactive cocktail of exosomes, enriched with specialized proteins, cytokines, growth factors, and microRNAs (miRNAs). These molecular components provide measurable evidence of their activation state, regenerative capabilities, and immunomodulatory precision.

[0189] This secretome is comprised of exosomes with a consistent particle size distribution, defined surface marker expression, and a tailored payload of miRNAs, as a result of the manufacturing process. The inclusion of keycytokines like TNF-a and SDF-1 during the activation process stimulates the MSCs to secrete a secretome.

[0190] Below is the detailed composition:

[0191] Protein Profile

[0192] Protein Concentration: Ranges from 0.7-43 pg / L across measured samples. Total Protein: Ranges from 4,760-38,578.8 pg.

[0193] RNA Profile

[0194] RNA Concentration: Ranges from 13.5-132 ng / pL.

[0195] Total RNA: Ranges from 64.0-2,632.0 ng.

[0196] Particle Profile

[0197] Protein Concentration: Ranges from 3.64-16.77 pg / L for samples 29-32. Particle Concentration: Ranges from 3.28 x 1010-1.51 x 1011particles / mL. Mean Particle Size: Ranges from 120.1-132.3 nm.

[0198] Mode Particle Size: Ranges from 70.9-90.2 nm.

[0199] Particle Number per pg Protein: Ranges from 4.8 x 107— 1.1 x 10®

[0200] Cell surface marker profile. The activation process yields MSCs with a distinctive biomarker profile, adhesion-related markers (CXCR4, CD29, CD44, CD49d, CD54, CD73, CD90, CD105, CD106), chemokine and cytokine receptor markers (CCR2, CCR7, CXCR3, CCR5, CX3CR1), homing and migration-enhancing markers (VLA-4, ALCAM, CD18, CD49e, MCAM), inflammation-responsive markers (TLR2, TLR4, HLA-G)

[0201] Exosomal Surface Markers

[0202] CD63 (%): Ranges from 0.00-15.21%.

[0203] CD9 (%): Ranges from 20.79-30.12%.

[0204] CD81 (%): Ranges from 22.99-51.90%.

[0205] Enriched MicroRNAs

[0206] RNA Concentration:

[0207] Ranges from 6.76-29.24 ng / pL.

[0208] miRNA Levels (Normalized Concentration):

[0209] miR-146b: Ranges from 0.74-1.19.

[0210] miR-24: Ranges from 0.74-1.19.

[0211] miR-124: Ranges from 0.74-1.17.miR-125: Ranges from 1.05-1.24.

[0212] miR-149: Ranges from 0.74-0.97.

[0213] miR-555: Ranges from 0.71-0.89.

[0214] Functional gene expression profile:

[0215] - TNFAIP6:

[0216] ACT (relative to GAPDH): Range from 6.594 to 8.328.

[0217] Fold Change (FC): Ranges from 1.158 to 3.327.

[0218] Conclusion. Demonstrates robust upregulation compared to regular MSCs with a fold change of up to 3.327, suggesting enhanced antiinflammatory activity.

[0219] - TGFB1:

[0220] CT (relative to GAPDH): Range from 2.361 to 4.649.

[0221] Fold Change (FC): Ranges from 1.060 to 4.883.

[0222] Conclusion: Fold change up to 4.883, compared to regular MSCs indicating strong pro-regenerative and anti-fibrotic signaling.

[0223] - VEGFA:

[0224] ACT (relative to GAPDH): Range from 3.692 to 5.240.

[0225] Fold Change (FC): Ranges from 1.148 to 2.924.

[0226] Conclusion: Upregulated up to 2.924-fold, compared to regular MSCs highlighting its contribution to angiogenesis and vascular repair. - COX2 / PTGS2:

[0227] ACT (relative to GAPDH): Range from 10.770 to 17.562.

[0228] Fold Change (FC): Ranges from 0.064 to 7.126.

[0229] Conclusion: Variable expression with a fold change of up to 7.126, compared to regular MSCs, associated with inflammation resolution pathways.

[0230] Functional Kynurenine Concentration profile (pg / mL):

[0231] Absorbance spectroscopy assay.

[0232] - Non-Activated Cells.

[0233] o Range: 0.38-0.49 pg / mL:

[0234] Passage 2: Passage average: 0.38 pg / mL

[0235] Passage 3: Passage average: 0.46 pg / mLPassage 4: Passage average: 0.48 pg / mL

[0236] Passage 5: Passage average: 0.49 pg / mL

[0237] - Activated Cells.

[0238] o Range: 9.01-16.71 pg / mL:

[0239] Passage 2: Passage average: 9.01 pg / mL

[0240] Passage 3: Passage average: 12.30 pg / mL

[0241] Passage 4: Passage average: 14.08 pg / mL

[0242] P Passage: Passage average: 16.71 pg / mL

[0243] HPLC assay.

[0244] - Non-Activated Cells:

[0245] o Range: 0.34-0.38 pg / mL

[0246] P2: Passage average: 0.37 pg / mL

[0247] P3: Passage average: 0.34 pg / mL

[0248] P4: Passage average: 0.34 pg / mL

[0249] P5: Passage average: 0.38 pg / mL

[0250] - Activated Cells:

[0251] o Range: 6.69-11.74 pg / mL

[0252] P2: Passage average: 6.69 pg / mL

[0253] P3: Passage average: 8.98 pg / mL

[0254] P4: Passage average: 10.31 pg / mL

[0255] P5: Passage average: 11.74 pg / mL

[0256] Example 7 - In vitro and ex vivo immunomodulatory effects of human placental mesenchymal stem cells in hidradenitis suppurativa

[0257] Study population and sample collection

[0258] Table 1. Demographic and clinical characteristics of patients with hidradenitis suppurativa and healthy controls.

[0259]

[0260]

[0261] SD, standard deviation; BMI, body mass index; IHS4, International Hidradenitis Suppurativa Severity Score System; CRP, C-reactive protein; PBMC, peripheral blood mononuclear cells.

[0262] Ten HS patients at Hurley stage II were included in the study. All patients had not been treated with systemic antibiotics for at least 6 weeks prior to enrolment. They were also biologically naive and did not have any other inflammatory disease. Patients exhibited at least moderate disease severity according to the IHS4 score. The clinical data indicate that our research cohort is representative of moderate or severe HS severity and relatively homogeneous [mean IHS4 score 8.5 (SD 2.2)] (Table 1). Four mm in diameter skin punch biopsies were taken from axillary HS lesional (inflammatory nodules) and HS perilesional (at a distance of >5 cm from visible HS inflammation area) skin areas (n=10). Three healthy control subjects (3 females, mean age 25 years, range 23-29 years) were included in the study, and 4 mm in diameter skin punch biopsies were taken from axillary healthy skin. Finally, the collection of blood samples from HS (n=3) and healthy (n=3) study subjects was conducted under aseptic conditions. Approval by theregional bio-ethics committee was obtained (Nr. BE-2-105), and each patient and control subject gave written informed consent.

[0263] Flow cytometry

[0264] PBMCs were isolated from HS patients and healthy volunteers using the method described by Oliver Vila et al. in 2018 (5). The isolation process involved gradient centrifugation to separate PBMC from venous blood, followed by staining with CFSE and stimulation with PHA. PBMCs were cultured in RPMI 1640 cell culture medium (Gibco) with 10% FBS. Additionally, patient PBMCs were co-cultured with either naive mesenchymal stem cells (n-MSCs), activated mesenchymal stem cells (a-MSCs), or high-dose 30 pg / ml adalimumab (HUMIRA®, AbbVie) to create the following groups: 1) n-MSCs, 2) a-MSCs, 3) adalimumab, 4) HS control and 5) healthy control. Concentrations of adalimumab were chosen according to previously published studies (6), and maximum concentrations were specified in the summary of product characteristics.

[0265] PBMCs were collected after 5 days and labeled with a panel of antibodies and stains: 7-AAD for viability, anti-CD3, anti-CD4, anti-CD8, anti-CD25 and antiCD^?. They were then subjected to flow cytometry analysis, which involved examination of 100,000 events per sample. The data obtained from the flow cytometry analysis were analyzed using FlowJo version 10.8.1 (BD, Ashland, Oregon, USA). The following populations were studied: live cells, CD3+ (lymphocytes), CD4+CD8- (T lymphocytes), CD4-CD8+ (Cytotoxic lymphocytes), CD4+CD25hiCD127lo (Treg lymphocytes). Overall proliferation of live cells as well as proliferation and relative counts of the above mentioned cell populations were studied. Normalized proliferation was calculated as described by Vila et al. (5). Briefly, absolute proliferation was calculated by subtracting the background proliferation in the non-stimulated condition from the proliferation in the stimulated condition. To assess the effect of coculture on proliferation, coculture absolute proliferation values were normalized to the absolute proliferation observed in stimulated single PBMC cultures.

[0266] Co-culture experimentSkin biopsy and MSC co-cultures were performed following the method described by Vossen et al. (7) with minor modifications. Biopsies were placed immediately after the procedure in punched-out 3 mm holes in the Transwell membrane (0.4 pm, celIQART, SABEll, Northeim, Germany) of a 12-well plate with the epidermis exposed to the air and the dermis immersed in 1 ml of DMEM (Sigma-Aldrich, St. Louis, MO) containing 0.5% heat-inactivated human AB serum (Sigma-Aldrich) and 0.1% of gentamycin (Gibco, Waltham, MA). 200,000 a-MSCs, n-MSCs (5.26x104 cells / cm2), or adalimumab (30 pg / ml) were added to the respective well resulting the following groups: 1) culture as negative control; 2) lesional control; 3) lesional a-MSCs; 4) lesional n-MSCs; 5) lesional adalimumab; 6) perilesional control; 7) perilesional a-MSCs; 8) perilesional n-MSCs; 9) perilesional adalimumab, 10) healthy individual control. Skin biopsies were incubated for 24 h at 37°C in an atmosphere of 5% CO2 and 98% humidity. Supernatants were centrifuged at 10,000 g for 1 min to remove cell debris and then stored at -80°C for later analysis.

[0267] Cytokine Luminex analysis

[0268] Cytokine protein concentrations in the supernatant were assessed using a custom-designed premixed Luminex Discovery assay (Bio-Techne, MN, USA) for the following cytokines: IL-1 (3, IL-10, IL-17, IFN-y and TNF-a. Values extrapolated from the standard curves were considered unreliable. Thus, a concentration = 0 pg / ml was assigned.

[0269] Statistical analysis

[0270] The results are presented as median values along with the interquartile range (minimum to maximum values). Statistical analysis was conducted using GraphPad Prism version 10.0.0 (GraphPad Software), considering a significant level of p<0.05. Group differences were assessed using the Kruskal-Wallis test, while Dunn’s test was applied for pairwise comparisons.

[0271] Results

[0272] Flow cytometry

[0273] Stimulation of PBMCs with PHA resulted in robust lymphocyte proliferation. CFSE dye was used, which is diluted as the cell proliferates. Comparingunstimulated (Figure 1A) and stimulated (Figure 1B) samples, changes in the CFSE histogram show dilution of CFSE, resulting from cell division. Four peaks are seen in the stimulated sample, corresponding to four generations of cells. We compared lymphocyte proliferation and relative changes in CD4, CD8 and Treg populations across different interventions. Both n-MSCs and a-MSCs inhibited overall lymphocytes proliferation (Figure 2, CD3 proliferation), as well as CD4 and Treg lymphocyte proliferation. Tregs n-MSCs resulted in a tendency for reduction, which did not reach significance. There were no significant differences in CD8 lymphocyte proliferation. Adalimumab did not show a significant effect on the overall lymphocyte proliferation.

[0274] Interestingly, when analyzing changes in CD4, CD8 or Treg distribution across different interventions, several significant results stood out (Figure 3). While both a-MSCs and n-MSCs reduced the proliferation of CD4 lymphocytes, neither intervention reduced the relative count. In fact, in the samples co-cultured with a-MSCs, we found a significant increase in CD4 lymphocyte percentage. Additionally, the amount of CD8 lymphocytes was greatly reduced in the overall lymphocyte population. As for Tregs, there were no significant differences across interventions.

[0275] Overall, these results show that a-MSCs and n-MSCs not only reduced lymphocyte proliferation but induced a shift in distribution, resulting in a greatly decreased amount of CD8 (cytotoxic lymphocytes), and an increase in CD4 (helper lymphocytes). While both a-MSCs and n-MSCs reduced Treg proliferation, this did not result in a reduced percentage of regulatory lymphocytes in the population, which are important for reducing inflammation. Therefore, in the samples with a-MSCs and n-MSCs, we can see a shift in balance towards a less cytotoxic lymphocyte population.

[0276] Cytokine Luminex analysis

[0277] Cytokine concentration in PBMC supernatants from HS patients and healthy individuals was analyzed. HS patient PBMCs were subjected either to regular cell culture medium (HS control) or co-cultured with n-MSCs, a-MSCs or adalimumab. They produced greater amounts of cytokines after stimulation (Figure 4), which shows an aberrant systemic immune response. Whencomparing cytokine concentrations across different interventions, we found that a-MSCs statistically significantly reduced pro-inflammatory cytokines (IL-17A, IFN-y) and anti-inflammatory IL-10 levels (Figure 4) when compared with HS control. Naive MSCs showed partial effects.

[0278] For the ex vivo model, we examined lesional and perilesional biopsies from HS patients and healthy individuals. HS patient biopsies were grown in regular medium (HS control) or co-cultured with n-MSCs, a-MSCs or adalimumab. The lesional control biopsies from HS patients showed significantly elevated IL1 -|3, IL-10 and IL-17A concentrations when compared with healthy controls (Figure 5). In perilesional biopsy samples, HS control patients’ results showed significantly elevated IL1-|3 and IL-10 concentrations when compared with healthy controls (Figure 6).

[0279] In lesional biopsies, n-MSCs showed a tendency to reduce inflammatory cytokines (IL-1 (3, IL-17A, TNF-a) and IL-10 concentrations when compared with the HS control group. Interestingly, a-MSCs statistically significantly increased IL-10 concentration when compared with HS control samples (Figure 5).

[0280] In perilesional biopsies, n-MSCs demonstrated reductions in pro-inflammatory cytokines (IL-1 (3, IL-17A) and stabilized IL-10 levels (Figure 6). Overall, these results show that the local immune response in HS patient skin is pro-inflammatory. Robust reduction of inflammation was seen in n-MSC samples, followed by a lesser reduction in a-MSC samples. Adalimumab also reduced inflammation, but it affected a smaller number of pro-inflammatory cytokines. To conclude, our findings further confirm that HS is driven by complex proinflammatory cytokine pathway dysregulation, as stated in previous studies of HS immunopathogenesis. Accordingly, MSC-based therapies are deemed a promising therapeutic intervention on account of their ability to modulate several immune pathways, thereby decreasing inflammation driven by HS.

[0281] References

[0282] 1. U.S. Pat. No. 5,486,359, “Human Mesenchymal Stem Cell”, published on January 23, 1996)2. U.S. Pat. No. 6,077,096, "Isolation, cryopreservation and therapeutic use of human amniotic epithelial cells", published 7 December 2000

[0283] 3. US Pat. No. 6,099,099, “Selective growth of target cell population”, published on January 15, 2002)

[0284] 4. U.S. Pat No. 6,022,743, “Three-dimensional culture of pancreatic parenchymal cells cultured living stromal tissue prepared in vitro”, published on February 8, 2000)

[0285] 5. Oliver-Vila I, Ramirez-Moncayo C, Grau-Vorster M, Marin-Gallen S, Caminal M, Vives J. Optimisation of a potency assay for the assessment of immunomodulative potential of clinical grade multipotent mesenchymal stromal cells. Cytotechnology. (2018) 70(1):31-44.

[0286] 6. Zouboulis CC, HouX, von Waldthausen H, Zouboulis KC, Hossini AM. HS 3D seboSkin model enables the preclinical exploration of therapeutic candidates for hidradenitis suppurativa / acne inversa. Pharmaceutics. (2023) 15(2):619

[0287] 7. Vossen ARJV, Ardon CB, van der Zee HH, Lubberts E, Prens EP. The anti inflammatory potency of biologies targeting tumour necrosis factor-a, interleukin (IL) 17A, IL-12 / 23 and CD20 in hidradenitis suppurativa: an ex vivo study. Br J Dermatol. (2019) 181:314-23

Claims

34Claims1 A method for producing a plurality of activated mesenchymal stem cells (MSC), the method comprising the steps:i) providing a sample comprising placenta tissue derived MSC, preferably obtained from the choriotrophoblastic region of the placenta;ii) initiating a cell culture comprising the MSC;iii) adding microscaffolds to the culture comprising the MSC;iv) allowing the MSC to adhere to the microscaffolds;v) culturing the MSC adhered to the microscaffolds in dynamic suspension in a bioreactor under hypoxic conditions for at least 6 days, preferably at least 7 days, more preferably at least 9 days, most preferably at least 11 days;vi) optionally repeating steps iii), iv) and v) at least once, preferably at least twice, more preferably twice;vii) culturing the MSC for at least 16 hours, preferably at least 20 hours, more preferably at least 24 hours in the presence of tumor necrosis factor-a, preferably in a concentration of 10 ng / ml, interferon-y, preferably in a concentration of 10 ng / ml, and stromal derived factor 1 , preferably in a concentration of 100 ng / ml, such that a plurality of activated MSC is obtained;viii) harvesting the plurality of activated MSC.

2. A method for producing a plurality of activated mesenchymal stem cells (MSC), the method comprising the steps:i) providing a sample comprising placenta tissue derived fibroblast-like cells, preferably obtained from the choriotrophoblastic region of the placenta;ii) culturing the placenta tissue derived fibroblast-like cells under hypoxic conditions in a 2D cell factory for at least 8 days in the presence of tumor necrosis factor-a, interferon-y, and stromal derived factor 1 , thereby obtaining MSC;iii) selecting and harvesting MSC from the 2D cell factory, wherein MSC that show spindle shaped morphology are selected;iv) initiating a cell culture comprising the selected MSC;v) adding microscaffolds to the culture comprising the MSC;35vi) allowing the MSC to adhere to the microscaffolds;vii) culturing the selected and harvested MSC in a bioreactor under hypoxic conditions, preferably at an oxygen concentration of between 1 - 5%, preferably of between 2 -4 %, more preferably of between 2 - 3%, most preferably of 2% for at least 6 days, preferably at least 7 days;viii culturing the MSC for at least 16 hours, preferably at least 20 hours, more preferably at least 24 hours in the presence of tumor necrosis factor-a, preferably in a concentration of 10 ng / ml, interferon-y, preferably in a concentration of 10 ng / ml, and stromal derived factor 1 , preferably in a concentration of 100 ng / ml, such that a plurality of activated MSC is obtained;;ix) harvesting the plurality of activated MSC obtained.

3. A method according to claim 1 , wherein the microscaffolds are cationic charged cross-linked poysterene microscaffolds, preferably cross-linked dextran or gelatinous-suspension microscaffolds.

4. A composition comprising a plurality of activated MSC, characterized in that the activated MSC express CD29, CD44, CD49d, CD54, CD73, CD90, CD105, CD106, VLA-4, ALCAM, CD18, CD49e, and MCAM, and expression of CXCR4, CCR2, CCR7, CXCR3, CCR5, CX3CR1, TLR2, TLR4, and HLA-G is increased by at least two-fold in the activated MSC when compared to MSC not cultured in the presence of tumor necrosis factor-a, preferably in a concentration of 10 ng / ml, interferon-y, preferably in a concentration of 10 ng / ml, and stromal derived factor 1 , preferably in a concentration of 100 ng / ml.

5. A composition comprising MSC-secreted exosomes, and optionally a plurality of MSC, characterized in at least 10%, preferably at least 15%, more preferably at least 20% of the MSC-secreted exosomes is positive for CD9 or CD81.

6. A composition according to claim 4 or 5, wherein the MSC express tumor necrosis factor alpha-induced protein 6 with a delta Ct relative to GAPDH of at least 4, preferably at least 5, more preferably at least 6, transforming growth factor beta 1 with a delta Ct relative to GAPDH of at least 2, vascular endothelial growth factor A with a delta Ct relative to GAPDH of atleast 2, prefarably at least 3, more preferably at least 3.5, and / or cyclooxygenas-2 with a delta CT relative to GAPDH of at least 5, preferably at least 7, more preferably at least 9, most preferably at least 10.

7. A composition according to any one of claims 4 -6, wherein less than 1 % of the MSC express or secrete SCF, FLT-3, ALDH X, or IL-6 and / or wherein the composition is essentially free of SCF, FLT-3, ALDH X, and IL-6.

8. A composition according to any one of claims 4 -7, obtainable by a method according to any one of claims 1 - 3.

9. A composition according to any one of claims 4 -8, for use as a medicament.

10. A composition for use according to claim 9, for treating an inflammory condition.

11. A composition for use according to claim 10, wherein the inflammatory condition is selected from the group consisting of acute inflammation, acute kidney injury, acute lung injury, acute liver injury, acute pancreatitis, acute severe colitis, acute nodular flares in hidradenitis suppurativa, an autoimmune disorder, and a chronic degenerative disease.

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