Composition enriched in mitochondria isolated from umbilical cord mesenchymal stem cells, useful in osteoarthritis

Abstract

The invention relates to a composition comprising mitochondria isolated from umbilical cord mesenchymal stem cells (UC-MSCs), which are specifically enriched and prepared for therapeutic use in osteoarthritis (OA). Mitochondria are isolated from UC-MSC cultures in earlier passages (P3- P7). The isolated mitochondria are suspended in a biocompatible carrier suitable for intra-articular injection. The invention further encompasses a method for treating and / or regenerating and / or ameliorating OA in a subject by administering the composition to the affected joint.

Description

[0001] Composition enriched in mitochondria isolated from umbilical cord mesenchymal stem cells, useful in osteoarthritis.

[0002] Description

[0003] Background of the invention

[0004] Osteoarthritis (OA) is a chronic condition of high prevalence and a main health burden contributing to global disability. It is also an independent risk factor for mortality.

[0005] Options for disease modifying treatments are lacking. Disease progression is driven by mechanical and proinflammatory stress, aging, and metabolic factors responsible for cartilage damage. Indeed, chondrocytes are the only cell type within mature cartilage, and several studies point to a mitochondrial bioenergetic failure of chondrocytes characterized by profound ATP depletion associated with disease. This leads to oxidative stress, further mitochondrial DNA damage, and increased rates of apoptosis and cartilage matrix degradation.

[0006] Recently, mitochondrial dynamics have been described as the main component that governs this process, such that increased mitochondrial fission leads to mitophagy and energetic failure, while fusion improves mitochondrial function and energy production, inhibiting chondrocyte apoptosis.

[0007] Due to the chondrogenic, anti-inflammatory, and regenerative properties of Mesenchymal stromal cells (MSCs), their therapeutic use in OA has been widely tested in Clinical trials by our group and others, and animal disease models.

[0008] Under metabolic stress, intercellular mitochondria transfer can restore bioenergetics and maintain the viability of recipient cells.

[0009] In the state of the art, the use of mitochondria extracted from mesenchymal cells to improve the function of other cells or tissues is known, but still in it's early stages. For example, the publication by the inventor Court, Angela C., et al. ("Mitochondrial transfer from MSCs to T cells induces Treg differentiation and restricts inflammatory response." EMBO reports 21.2 (2020): e48052.), discloses the use of mitochondria from mesenchymal cells to transform T cells and induce Treg differentiation. Where this transformation of T cells was performed in vitro. We are not aware of any prior state of the art that has considered the use of a composition enriched in UC MSC mitochondria for in vivo treatment of OA in a subject's joint, as proposed in the present invention.

[0010] As we indicate, there are presently no disease modifying treatments for OA, a disease with great social and clinical impact. The recent description of the tissue and cell regenerating properties of the transfer of MSC-derived mitochondria (MitoT) open a "window of opportunity" to intervene the OA process. Given the central role of MT in the energetic failure of OA, the inventors sought to assess 1) if human umbilical cord-derived MSCs (UC-MSCs) would transfer their MT to human chondrocytes and 2) if MitoT could restore some of the dysfunctional traits of the OA chondrocyte, including metabolic activity, ATP production, mitochondrial dynamics and oxidative stress management.

[0011] The results were positive, inventors found evidence for both phenomena, and they proceeded then to demonstrate that the intra-articular injection of UC-MSC led to the improvement of disease in an animal model of OA. These findings represent a new therapeutic strategy in the treatment of OA.

[0012] Figures

[0013] Figure 1. A-B) Western Blot analysis of proteins related to the mitochondrial fusion / fission process in MSC-MT mitocepted OA-Ch. (A) Representative blots at 24, 48 and 72 hours post mitoception and (B) average bar graphs from three different OA patient samples. The graph depicts fold expression of tested proteins in MitoT+ OA cells relative to No MitoT cells, which are set at 1. C) Quantitative analysis of mitochondrial morphology in MitoT+ OA-Chs after mitoception with MSC- MT, compared to no-mitocepted controls (No MitoT), assessed by confocal microscopy (n=10-38 independent cells analyzed per group).

[0014] Figure 2. A) Fold change of SOD2 protein expression levels by Western blot analysis in MitoT+ OA- Chs after 48 hours post-mitoception with MSC-MT (n=3). B) Mitochondrial superoxide dismutase activity levels (MnSOD) in MitoT+OA-Chs and non-mitocepted Chs, after 24 hours post-mitoception with MSC-MT (n=4). C) Average of the % H2DCFDA+ (ROS levels) population on MitoT+ OA-Chs compared to control No MitoT after incubation with 25 pM MD, by flow cytometry analysis (n=4). D) Percentage of live cells on MitoT+ OA-Chs after 24 hours incubation with increasing concentrations of hydrogen peroxide (H2O2) compared to No MitoT control (n=4). E) Percentage of TUNEL-positive cells of MitoT+ OA-Chs after 15 minutes incubation with 25 pM menadione, compared to No MitoT control, calculated from four independent confocal images per group (120- 180 total cells analyzed).

[0015] Figure 3. Human MT detection in mouse chondrocytes isolated from articular cartilage.

[0016] (A) In vivo experimental design of intra-articular injections with MSC-derived MT to evaluate its integration in the mouse joint. (B) qPCR analysis of human 02-microglobulin (B2M), human specific mitochondrial (MT) and mouse specific (b-actin) gene expression levels in chondrocytes isolated from MT-treated mice, no treated mice or human chondrocytes as control (n = 2). E = not detected.

[0017] Figure 4. A) Representative mice images following intraarticular injections with Mitoview-labeled MSC-mitochondria in CIOA mice (white arrows), evaluated immediately after treatment, by Odyssey CLx Imager. Equal volume of sodium chloride (NaCI) was used in control OA mice. B) Relative intensity density quantification after 0, 1, 24, 48 and 72 hours post-treatment with with Mitoview- labeled MSC-mitochondria in CIOA mice. Figure 5. Therapeutic effect of UC-MSC derived-MT treatment in a preclinical model of OA.

[0018] (A) In vivo experimental design of collagenase-induced model of OA (CIOA), treated isolated human MSC derived mitochondria (MSC-MT). (B) Bone mineral density analysis of CIOA mice (OA), CIOA mice transplanted intraarticularly with isolated MT derived from 2xl05MSCs (OA+MT) or control group (sham, contralateral leg injected with sodium chloride). Average of mineralization levels for each joint zone (bottom panel) (n = 2 experimental replicates, with at least 8 mice per group). (C) OA damage score quantification in knee joint sections obtained from CIOA mice (OA), CIOA mice transplanted intraarticularly with isolated MT derived from 2xl05MSCs (OA+MT) or control group (sham), for each joint zone (n = 2 experimental replicates, with at least 8 mice per group). Graphs show mean ± SEM and statistical analysis by non-parametric Mann-Whitney U test (*** p < 0.001; ** p < 0.01; * p < 0.05).

[0019] Figure 6. UC-MSC derived-MT restores cartilage damage in a preclinical model of OA. (A) In vivo experimental design of collagenase-induced model of OA (CIOA) treated with isolated human MSC derived mitochondria (MSC-MT), to evaluate cartilage damage by histological knee sections. (B) Collagen density quantification calculated from two independent images per group from articular knee cartilage sections obtained from CIOA mice (OA), CIOA mice transplanted intraarticularly with isolated MT derived from 2xl05MSCs (OA+MT) or control group (sham) stained with Masson's trichrome (n =1 experimental replicate, with 3 mice per group). (D) Representative confocal microscopy of TUNEL staining (green) in articular knee cartilage sections obtained from CIOA mice (OA), CIOA mice transplanted intraarticularly with isolated MT derived from 2xl05MSCs (OA+MT) or control group (sham). Cell nucleus were stained with DAPI (blue). (lOx). (E) Quantification of the number of TUNEL-positive cells per unit area (500.000 pm) in the cartilage region (ROI) from the articular knee sections obtained from CIOA mice (OA), CIOA mice transplanted intraarticularly with isolated MT derived from 2xl05MSCs (OA+MT) or control group (sham) (n = 1 experimental replicate, with 3 mice per group). Description

[0020] The invention relates to a composition comprising mitochondria isolated from umbilical cord mesenchymal stem cells (UC-MSCs), which are specifically enriched and prepared for therapeutic use in osteoarthritis (OA). Mitochondria are isolated from UC-MSC cultures in earlier passages (P3- P7). The isolated mitochondria are suspended in a biocompatible carrier suitable for intra-articular injection. The invention further encompasses a method for treating and / or regenerating and / or ameliorating OA in a subject by administering the composition to the affected joint.

[0021] The UC-MSCs used in the invention are derived from umbilical cord tissue using standard isolation and culture techniques. UC-MSCs are cultured under controlled conditions until reaching an early passage stage, specifically passages 3 to 7 (P3-P7). Early passage cells are selected to ensure optimal mitochondrial quality and therapeutic efficacy.

[0022] The mitochondria are isolated from UC-MSCs using a differential centrifugation process. Specifically:

[0023] - A cell pellet is prepared containing 1 to 10 x 106UC-MSCs.

[0024] - The cells are lysed to release intracellular components, and sequential centrifugation steps are performed to separate mitochondria from other cellular debris.

[0025] - The isolated mitochondria are washed and concentrated into a sterile preparation for therapeutic use.

[0026] The isolated mitochondria are suspended in a biocompatible carrier suitable for intra-articular injection. The preferred carrier is a cell culture medium optimized for mammalian cells, ensuring mitochondrial viability and compatibility with the joint environment. The carrier medium may be supplemented with stabilizers to preserve mitochondrial functionality during storage and administration.

[0027] In one embodiment, the invention also provides a method for treating and / or regenerating and / or ameliorating OA in a human subject. The method involves intra-articular administration of a therapeutically effective amount of mitochondria-enriched composition directly to the affected joint. - The injection is performed using standard medical techniques under aseptic conditions.

[0028] As is shown in the examples the inventors found that the therapeutic effects of the composition enriched in mitochondria may be attributed to a restoration of cellular energy balance in the affected joint. Also the healthy mitochondria help in the modulation of inflammation through mitochondrial immunomodulatory properties. And contributes to the stimulation of cartilage regeneration and reduction of oxidative stress.

[0029] This invention provides a novel therapeutic approach for OA by leveraging the regenerative and immunomodulatory properties of mitochondria isolated from UC-MSCs. The detailed processes and applications described herein demonstrate the potential of this composition as a transformative treatment for osteoarthritis.

[0030] In one embodiment the invention is directed to a composition enriched in mitochondria, isolated from umbilical cord mesenchymal stem cells (UC-MSC), for use in the treatment or amelioration of osteoarthritis (OA) in the joint of a subject. Wherein the mitochondria are isolated using a process comprising differential centrifugation from a pellet containing 1 to 10 xlO6UC-MSC from early passages, i.e. passage 3 to passage 7, from a UC-MSCs culture. In the composition the mitochondria are suspended in a biocompatible carrier suitable for intra-articular injection. Preferably, the carrier is a media for the culture of mammal cells.

[0031] Examples of media for the culture of mammal cells useful as carriers are DMEM, Ringer's lactate, among others xenofree media.

[0032] In a second embodiment the invention is directed to a method to obtain the composition described wherein the method comprises a) providing a culture of umbilical cord mesenchymal cells in passages 3 to 7; b) washing the cells and extracting the mitochondria with a process comprising differential centrifugation from a pellet containing 1 to 10 xlO6UC-MSC c) suspending the extracted mitochondria in a media for the culture of mammalian cells, to form a composition enriched in mitochondria isolated from UC-MSC. Also, in a third embodiment the invention is also directed to a method for treating and / or regenerating and / or ameliorating osteoarthritis in a subject, comprising the administration of a therapeutically effective amount of a composition enriched in mitochondria isolated from umbilical cord mesenchymal stem cells, as described above, to the affected joint of a subject.

[0033] The invention could be best understood in the light of the next examples.

[0034] Examples

[0035] Example 1. Isolation of mitochondria from UC-MSC.

[0036] Umbilical cord (UC) derived MSCs were isolated from healthy donor umbilical cords and characterized according to the International Society for Cell and Gene Therapy (ISCT) criteria, under GMP conditions. All experiments were performed using early passage (P3-P7) UC-MSCs cultured in the culture media Dulbecco's Modified Eagle's Medium DMEM supplemented with 10% Fetal Bovine Serum, 1 mM L-glutamine and 1% Penicillin / Streptomycin (P / S). All cells were grown in humidified incubation chambers at 37 °C with 5% CO2.

[0037] UC-MSC MT was isolated from previously MitoTracker labeled donor cells using the Mitochondria Isolation Kit (Thermo Fisher Scientific), following manufacturer's instructions. Briefly, cultures were dissociated to lift cells from the plate, washed, and centrifuged (2 minutes at 1500 g). Pellets containing 1 to 10xl06UC-MSC were homogenized with 200 pL of reagent A from the Kit and 2 pL of protease inhibitor (Halt Protease and Phosphatase Inhibitor Cocktail, EDTA-Free, Thermo Scientific) by vortexing for 5 seconds. Then, the culture was incubated on ice for 2 minutes. Subsequently, 2.5 pL of reagent B from the Kit was added, and the culture was homogenized by vortexing for 10 seconds every 30 seconds for 5 minutes, with incubation on ice in between. Then, 200 pL of reagent C from the Kit and 2 pL of the protease inhibitor were added. The contents were mixed by gently inverting the tube for 30 seconds. The tube was centrifuged at 700 g for 10 minutes and the supernatant was transferred to a new sterile 1.5 mL Eppendorff tube. This new tube was centrifuged at 3,000 g for 15 minutes. Finally, the supernatant (cytosolic fraction) was discarded, and the pellet (mitochondrial fraction) was resuspended in MSC culture media were maintained on ice until artificial transfer (within 2-3 hours). MT isolated from 1 to 10xl06MSCs and resuspended in MSC culture media (DMEM) were maintained on ice until artificial transfer of mitochondria (MitoT), within 2-3 hours.

[0038] Example 2. MitoT promotes MT fusion, restoring MT dynamics in the OA chondrocyte. Mitochondria isolation and artificial transfer (Mitoception).

[0039] Mitoception was performed on primary chondrocytes seeded the day before (100.000 chondrocytes per well on a 12-well plate), using the amount of MSC MT corresponding to MSC:Ch ratios of 1:10, 1:5 and 1:1. Oligomycin A (Sigma-Aldrich) was added at 1 ug / mL as control of ATP productive active- MT. For each sample, 50 pL of CellTiter-Glo® Reagent was added and the plate was incubated for 30 min at room temperature, followed by a luminescence reading using a BioTek FLx800 microplate reader.

[0040] The following day cells were washed with PBS IX and used for experimental procedures.

[0041] We assessed the changes in mitochondrial dynamics within the OA-Chs in response to MitoT. We evaluated the expression of the main fusion / fission regulator proteins at days 1-3 post mitoception. Quantitation of the main fusion / fission regulator proteins was performed by Western blot using Antibodies against Mitofusin-2 (MFN2), fusion marker, and antibodies that recognize phosphorylated Ser616-DRP1 (p-DRPl) and DRP1, to determine the (p-DRPl) / DRPl ratio, marker of MT fission.

[0042] To the Western Blot analysis primary human chondrocyte cells were collected after 24, 48 or 72 hours post-mitoception with MSCs-MT and resuspended with 50 pl of cold RIPA buffer containing protease / phosphatase inhibitors (Cell Signaling Technology, Danvers, MA, USA). Samples were then sonicated for 5 min at high intensity and centrifuged at 13200 rpm for 15 min at 4°C to discard cellular debris. Supernatants containing proteins were collected and protein quantification was performed using the Bradford method following the manufacturer's instructions (Bio-Rad, Hercules, CA, USA). 20-30 pg of total protein were loaded and run in 10% SDS-PAGE gels. 5% of BSA in PBS- Tween 0,01% was used for blocking and primary antibodies were used in a dilution of 1:1000 prepared in PBS-Tween 0,01%, overnight with agitation at 4°C. Secondary fluorescent antibodies were prepared in a 1:20000 dilution of 5% BSA in PBS-Tween (0,01%) (secondary antibodies from Invitrogen: a-mouse Alexa Fluor 680 #A32729 and a-rabbit Alexa Fluor 800 #A32735). Finally, fluorescence was detected in the Odyssey®CLx and analyzed with Image Studio Lite software (version 5.2).

[0043] At 24 hours, we detected a change in the level of expression of proteins related to mitochondrial fusion, with a significant increase in Mitofusin-2 (MFN2) (p<0.01) compared to No MitoT OA-Chs (Figure 1A-B). Conversely, the level of Ser616 phosphorylated Dynamin-related protein 1 (p-DRPl) - that promotes the MT fission process- was significantly decreased (p<0.05) in the MitoT+ OA-Chs (Figure 1A-B). significatively increased MT perimeter and MT area in MitoT+ OA-Chs (Figure 1C), indicate that MitoT promotes increased levels of MT fusion, restoring one of the main aspects of the unbalanced mitochondrial dynamics reported in the OA chondrocyte.

[0044] Example 3. MitoT grants oxidative stress resistance to OA chondrocytes, entailing anti-apoptotic effects.

[0045] We observed a 1.8-fold increase in SOD2 protein expression levels was observed in three different MitoT+ OA chondrocyte primary culture samples compared to No MitoT (p=0.012) (Figures 2A). Moreover, we observed a significant 2.9-fold increase in mitochondrial SOD activity levels (MnSOD) in MitoT+ OA-Chs compared to No MitoT cells (p=0.03) (Figure 2B). We measured ROS by flow cytometry, with the fluorogenic H2DCFDA reagent. Our results showed that MitoT+ OA-Chs had decreased percentage of ROS+ population compared to No MitoT Chs, when incubated with menadione (p=0.022) (Figure 2C).

[0046] In order to evaluate whether increased SOD2 by MitoT confers protection against cell death caused by increased oxidative stress in OA, we evaluated cell viability in MitoT+ OA-Chs using Annexin V / PI assay by flow cytometry. After 24 hours post-mitoception, oxidative stress was induced with increasing concentrations of hydrogen peroxide (H2O2). A significant 4-fold increase in cell viability was observed in MitoT+ OA-Chs treated with 250 pM of H2O2 for 24 hours, compared to No MitoT Chs (p=0.03) (Figure 2D). Confocal imaging of apoptotic cell death also confirmed the decrease (p = 0.043) in TUNEL-positive cells in OA-Chs after MSC-derived MitoT (Figure 2E). Taken together, these results demonstrate the anti-apoptotic role against oxidative stress that MitoT from UC-MSC confers to OA-Chs in vitro. Example 4. Therapeutic effect of intra-articular injection of UC-MSC derived MT in a preclinical model of OA.

[0047] The significant effects on the OA-Ch triggered by MitoT in vitro led us to explore the translational potential of the intra-articular injection of UC-MSC-derived MT in a mouse model of OA (Figure 3A). qPCR confirm the detection of the human mitochondrial gene within the isolated murine Chs harvested from MT-treated knee joints (Figures 3B). We determined the biodistribution of MSC- derived MT in CIOA murine model. Between 24 and 48 hrs after intraarticular injection MT were detected in the knee (Figure 4A-B).

[0048] Functional MSC-derived MT were isolated and injected intra-articularly in the knee joints of mice with collagenase-induced knee OA (Figure 5A). We assessed the effect of MitoT on histomorphometric bone parameters by micro-computed tomography (MicroCT - pCT) and histopathological analysis. At day 42, both OA knee joints and sham non-treated knees were collected for imaging and histopathology (Figure 5A). We evaluated bone parameters in four sections of each knee. While pCT images revealed the characteristic increase in knee bone mineralization in the OA mice groups, joints receiving MSC-derived MT injections (OA+MT group) exhibited a significant reduction (p < 0.01) in the bone mineral density (Figure 5B), that indicates less disease severity. Histological analysis of all knee sections also showed a significant decrease (p < 0.05) in the histological cartilage damage scores in the OA+MT group compared to the untreated OA mice (Figure SC).

[0049] Preliminary results showed that collagen of the cartilage matrix was increased in mice after 7 days post intra-articular injections of isolated MSC-MT (OA+MSC) compared to the untreated OA mice (Figures 6A-C). Furthermore, we observed that chondrocyte apoptosis induced in the OA knee joints tends to decrease after MT treatment.

[0050] These results point to a therapeutic effect of MitoT that is compatible with the restoration of MT homeostasis in the diseased chondrocyte, leading to cartilage regenerating effect in-vivo.

Claims

CLAIMS1.- A composition enriched in mitochondria, isolated from umbilical cord mesenchymal stem cells (UC-MSC), for use in the treatment or amelioration of osteoarthritis (OA) in the joint of a subject.2.- The composition of Claim 1, wherein the mitochondria are isolated using a process comprising differential centrifugation from a pellet containing 1 to 10 xlO6UC-MSC from early passages, i.e. passage 3 to passage 7, from a UC-MSCs culture.3.- The composition of Claim 1, wherein the mitochondria are suspended in a biocompatible carrier suitable for intra-articular injection.4.- The composition of Claim 3, wherein the carrier is a media for the culture of mammal cells.5.- A method for treating and / or regenerating and / or ameliorating osteoarthritis in a subject, comprising the administration of a therapeutically effective amount of a composition enriched in mitochondria isolated from umbilical cord mesenchymal stem cells, according to claim 1, to the affected joint of a subject.

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